Process for the production of 2-[4-(3- and 2-fluorobenzyloxy) benzylamino] propanamides

ABSTRACT

A process for obtaining therapeutically active 2-[4-(3- and 2-(fluorobenzyloxy)benzylamino]propanamides and their salts with pharmaceutically acceptable acids with high purity degree, in particular, with a content of dibenzyl derivatives impurities lower than 0.03%, preferably lower than 0.01% by weight. 
     The process is carried out by submitting the Schiff bases intermediates 2-[4-(3- and 2-fluorobenzyloxy)benzylideneamino]propanamides to catalytic hydrogenation in the presence of a heterogeneous catalyst in a protic organic solvent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is Continuation-In-Part application of International ApplicationNo. PCT/EP2007/005105, filed Jun. 8, 2007, designating the U.S., whichclaims the benefit of European patent application no. 06012565.5, filedJun. 19, 2006, the disclosures of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a new process for the production of(S)-2-[4-(3-fluorobenzyloxy)-benzylamino]propanamide, i.e. safinamide(Ia) and (S)-2-[4-(2-fluorobenzyloxy)-benzylamino]propanamide, i.e.ralfinamide (Ib) and their salts, in high yields and very highenantiomeric and chemical purity. This method is also very useful fortheir production in large quantites.

BACKGROUND

Safinamide (NW-1015, FCE-26743A, PNU-151774E) is a sodium channelblocker, a calcium channel modulator, a monoamino oxidase B (MAO-B)inhibitor, a glutamate releasing inhibitor and a dopamine metabolismmodulator.

Safinamide is useful in the treatment of CNS disorders, in particular ofepilepsy, Parkinson's disease, Alzheimer's disease, depression, restlesslegs syndrome and migraine (WO 90/14334, WO 04/089353, WO 05/102300, WO04/062655).

Ralfinamide (NW-1029, FCE-26742A, PNU-0154339E) is a sodium channelblocker useful in the treatment of pain conditions, including chronicpain and neuropathic pain, migraine, bipolar disorders, depressions,cardiovascular, inflammatory, urogenital, metabolic and gastrointestinaldisorders (WO 99/35125, WO 03/020273, WO 04/062655, WO 05/018627, WO05/070405, WO05/102300, WO 06/027052).

SUMMARY

It has now been discovered that the large scale preparations ofsafinamide and ralfinamide according to the methods described in theprior art, contain two undesired impurities, i.e., respectively,(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamide(IIa) and(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamide(IIb), and their salt, in particular the respective methanesulfonates(IIc) and (IId)

This fact is of particular relevance because of the very high toxicityof the two impurities named above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides total locomotor activity over 30 min. Mice were treatedwith ralfinamide (10; 30 and 60 mg/kg, po); or saline and returned totheir home cage. After 30 min they received an injection of Amph (2.5mg/kg; ip); Amph/CDP mixture (2.5 and 3.125 mg/kg; ip) or salineimmediately prior to be placed in the test cage. Locomotor activity wasrecorded for 30 min. Two-way ANOVA shows a main effect of Amph treatment(p<0.0001) and of ralfinamide treatment (p=0.0002) but not aninteraction of the two (p=0.202). Bonferroni's multiple comparison testsshows a statistical difference of ralfinamide 30 and 60 versus Amph/CDPmixture; and for ralfinamide 60 versus Amph alone ** P<0.01; ***P<0.001.n=12 mice per group.

DETAILED DESCRIPTION

Many of the drug candidates fail in clinical trials because ofunforeseen effects on human metabolism, or toxicity, due to unwantedimpurities and, therefore, the elimination of such impurities in earlypre-clinical phase is important and strongly desirable.

At preclinical level, the “drugability” of new compounds can be assessedusing a very well established battery of in vitro assays, such asinteraction with drug-metabolizing enzymes, cytotoxicity, metabolicstability and profiling, membrane permeability, intrinsic clearance andhuman ether-a-go-go related gene (HERG) channel blockade etc.

The Cytochrome P450 (CYP 450) system is the principal enzyme system forthe metabolism of lipophilic xenobiotics, including drugs, carcinogens,and environmental pollutants. CYP 450 is a heme-containing, membranebound, multienzyme system that is present in many tissues but is presentat the highest level in liver. In human liver, it is estimated thatthere are 15 to 20 different xenobiotic-metabolizing CYP 450 forms. Sofar, more than fourteen CYP gene families have been identified inmammals. Despite the existing high homology, extensive studies haverevealed that each CYP family and subfamily has distinct roles inxenobiotic metabolism. Three CYP families CYP1, CYP2 and CYP3 accountfor about 70% of human hepatic microsomes CYPs with CYP3 accounting forapproximately 30%. These CYPs are the major responsible for themetabolism of most marketed drugs.

The CYP1 family contains several members that include CYP1A1, CYP1A2 andCYP1B1 and they are involved in the metabolism of acetaminophen,clomipramine and imipramine.

The CYP2 family contains several subfamilies including CYP2A, CYP2B,CYP2C, CYP2D and CYP2E. The CYP2C subfamily contains at least sevenmembers. CYP2C9 is responsible for the metabolism of ibuprofen,diclofenac, tolbutamide and torsemide. CYP2C19 is the major isoenzymemetabolizing diazepam and omeprazole. CYP2D6 has been shown to beresponsible for metabolizing over 30% of the drugs on the market,including, antidepressants and cardiovascular and anti-psychotic drugs.

In the CYP3 family, three isoforms have been identified in human liver.Human CYP3A4 has been recognized to be the most important isoform indrug metabolism. To date, metabolism catalyzed by CYP3A4 is the majorelimination route for nearly 50% of marketed drugs.

Because of their importance in drug metabolism, both CYP3A4 and CYP2D6are often involved in drug-drug interactions and several clinically usedcompounds have been identified as potent inhibitor of these CYP 450isoforms such as ketoconazole, terfenadine, erythromycin, miconazolepropanolol and quinidine, respectively. This imposes a clear limitationon the use of these drugs.

A further problem, consisting in sudden death as a side effect of theaction of non antiarrhytmic drugs, is a major pharmacological safetyconcern facing the pharmaceutical industry and the health regulatoryauthorities. In recent years, at least five blockbusters drugs(astemizole, sertindole, terfenadine, cisapride, grepafloxacin) havebeen withdrawn from the market due to reports of sudden death. In allcases, long QT Syndrome (LQTS), an abnormality of cardiac musclerepolarization, that is characterized by the prolongation of the QTinterval in the electrocardiogram, was implicated as a predisposingfactor for “torsades de pointes”, a polymorphic ventricular tachycardiathat can spontaneously degenerate to ventricular fibrillation and causesudden death. Congenital LQTS can be traced back to several possiblemutations resulting in defects in sodium channels, and two differentpotassium channels: the rapidly activating delayed rectifier (I_(Kr))and the slowly activating delayed rectifier (I_(Ks)). Importantly,virtually every case of a prolonged duration of cardiac action potentialrelated to drug exposure (acquired LQTS) can be traced to one specificmechanism: blockade of I_(Kr), current in the heart. This current, amajor contributor to phase 3 repolarization at the end of QT interval,is conducted by tetrameric pores, with the individual subunits encodedby HERG. With blockade of HERG K⁺ channels widely regarded as thepredominant cause of drug-induced QT prolongation, early detection ofcompounds with this undesirable side effect has become an importantobjective in the pharmaceutical industry.

Compounds with strong inhibition of drug-metabolizing enzymes, inparticular CYP 450 enzymes, and HERG channel blocking properties have ahigh probability to be toxic and that their development has to bestopped at an early-stage.

As shown in the Table 1 the impurities (IIa and IIb), as themethanesulfonate salt (IIc and IId), strongly inhibit in the micro- andsubmicro-molar range CYP3A4, CYP2D6, CYP2 C19, CYP2 C9 and HERG currentsand are highly cytotoxic, compared with safinamide methanesulfonate (Ic)and ralfinamide methanesulfonate (Id) with high purity degrees,synthetized using the process of this invention.

TABLE 1 HERG Cytotoxicity CYP3A4 CYP2D6 CYP2C19 CYP2C9 CYP1A2 CompoundIC₅₀, μM IC₅₀, μM IC₅₀, μM IC₅₀, μM IC₅₀, μM IC₅₀, μM IC₅₀, μM Impurity1.20 6.70 0.05 0.77 0.42 7.29 >40 IIc Safinamide 27.0 248.0 >40 >4023.85 >40 >40 methanesulfonate Impurity 2.66 15.00 0.05 0.92 1.898.01 >40 Iid Ralfinamide 18.0 >300 >40 >40 >40 >40 >40 methanesulfonate

Table 2 shows comparative results (IC₅₀) about the inhibition of thecytocrome CYP3A4 using highly pure safinamide and ralfinamidemethanesulfonate, synthesized using the new process of this invention,with safinamide and ralfinamide obtained with the same process in thepresence of 0.3% of the impurity IIc and IId, respectively.

When 0.3% of the impurities IIc and IId are added to highly puresafinamide and ralfinamide methanesulfonate, a significant decrease inIC₅₀ on CYP3A4 is observed in both cases meaning that the impuritiescontribute to a strong inhibition of the enzyme activity.

TABLE 2 CYP3A4 Compound IC₅₀, μM Safinamide methanesulfonate >40Safinamide methanesulfonate plus 18 0.3% IIc impurity Ralfinamidemethanesulfonate >40 Ralfinamide methanesulfonate plus 0.3% 7.76 IIdimpurity

As shown in Table 3 the impurity (IIc) increases, starting from 3 mg/kgip, the mortality in the mice Maximal Electroshock (MES) test withoutany pharmacological activity, i.e. protection from convulsions.

TABLE 3 MES 3 mg/kg ip 10 mg/kg ip 30 mg/kg ip % pro- dead/ % pro- dead/% pro- dead/ Compound tection live tection live tection live Safinamide50 0/10 100 0/10 100 0/10 methanesulfonate Impurity IIc  0 5/10  0 4/10 0 4/10

Table 4 reports that the impurity IId, when given p.o. at 10 and 20mg/kg, in the Maximal Electroshock test (MES) doesn't protect mice fromconvulsions if compared with the same doses of ralfinamidemethanesulfonate.

TABLE 4 MES 10 mg/kg p.o. 20 mg/kg p.o. Protection Dead/ Dead/ Compound% live live Ralfinamide 60% 0/10 90% 0/10 methanesulfonate Impurity IId 0% 0/10  0% 0/10

Based on all these data, the impurities IIc and IId, present insafinamide and ralfinamide, respectively, synthesized with the processdescribed in WO 90/14334 and by Pevarello et al in J. Med. Chem, 1998,41, 579-590 show in vitro some undesirable features, such as cellulartoxicity, strong inhibition of some isoform of CYP 450, HERG channelblockade and no protective activity in an “in vivo” model of epilepsy.

One of the important aspects of CYP is the variation among differentpopulation groups. Variations in drug metabolism are of great importancein clinical studies. Considerable variation in the enzymatic activity ofCYP3A4 and CYP2D6 has been demonstrated between different ethnic groupsand even among different individuals in the same ethnic group. Thedifference in the CYP activity among individuals varies significantly,depending upon different isoenzymes. Changes in the CYP expression levelof different individuals can cause variations in drug metabolism. Moreimportantly, polymorphism can also result in CYP enzyme variants withlower or higher enymatic activity that leads to variations in drugmetabolism. CYP2D6 polymorphism is a well-studied topic in drugmetabolism. In clinical studies, pronounced variations betweenindividuals was first found in the metabolism of antihypertensive andantiepileptic drugs. Elimination of CYP2D6 metabolized drugs is slowerin those individuals who carry defective CYP2D6 alleles. Individualswith slow metabolism are classified as poor metabolizers (PM), whilecatalytically competent individuals are called extensive metabolizers(EM): The incidence of the PM phenotype in population of differentracial origin varies: approximately 5 to 10% of Caucasians are of the PMphenotype, but only 1% in Asian population. CYP2C19 is another importantpolymorphic isoform that has clinical implications.

Taken into account these observations, a compound that does notinterfere with CYP450 isoforms (neither inhibition nor induction) has avery low risk for drug-drug interactions in clinical practice and can besimply and safely prescribed by physicians.

In particular, drugs that not interfere with the cytochromes of theCYP450 system are particularly indicated for the therapeutical treatmentof individuals that are classified as poor metabolizers (PM) or for thetherapeutical treatment of patients who are concomitantly consumingother drugs which are known to interact with said cytochromes, such asketoconazole, terfenadine, erythromycin, miconazole, propanolol andquinidine, and/or are known to have HERG channel blocking properties.

Bipolar disorders are debilitating psychiatric illnesses characterisedby episodes of depression and mania, which often alternate and increasein severity and frequency over time. Bipolar disorders are not easy torecognize because many symptoms overlap with other psychiatricdisorders. Exact cause is unknown, but changes in the level of brainneurotransmitters, and psychosocial factors may be involved.

The pharmacology treatments so far available are various and withdifferent mechanisms of action. The drugs currently used in clinicalpractice range from lithium to other substances such as valproate,carbamazepine and lamotrigine.

For most of these drugs the mechanisms of action at the molecular levelsare not completely understood, and current knowledge indicates that mostof these drugs have more than one mechanism of action, each of which maycontribute to therapeutic efficacy to a variable extent depending on thecondition being treated. It would seem likely that the clinical efficacyof these drugs in the treatment of psychiatric disorders is related tomultifactorial mechanisms. It is known that not all anticonvulsants areactive in bipolar disorders as for example gabapentin (Frye at al, J.Clinical. Pharmacol.; 2000 20: 607-614; Pande et al, Bipolar Disord.;2000 2: 249-55), levetiracetam (Grunze et al, J. Clin. Psychiatry.; 200364: 781-4) and tiagabine (Yatham et al, J. Clin. Psychiatry.; 200465(suppl.10): 28-35). Therefore, the fact that compounds of the presentinvention show antiepileptic activity in some models (WO90/14334) doesnot necessarily imply activity in bipolar disorders. According to thecommon clinical practice, safinamide and ralfinamide methanesulfonates(Ic and Id) are usually administered to the patient in need thereof fora long period of time, subdivided in several daily dose. This isparticularly the case of therapeutical applications wherein the diseaseto be treated is: Parkinson's disease, Alzheimer's disease and restlesslegs syndrome (for the use of Safinamide) or chronic or neuropathicpain, cardiovascular or inflammatory disorders (for the use ofRalfinamide). Although the daily dosage may vary according to thespecific conditions and needs of the patients, the safinamidemethanesulfonate daily dosage may usually range from 10 mg/day to 800mg/day, while ralfinamide methanesulfonates daily dosage may usuallyrange from 10 mg/day to 1 g/day. Under these conditions, and inconsideration of the data reported above, it is highly advisable to keepthe level of the impurities (IIa) and (IIb) or the salts thereof, inparticular the methanesulfonate salts (IIc) and (IId) in thepharmaceutical dosage forms of safinamide and ralfinamide or the saltsthereof as low as possible, in any case lower than 0.03%, preferablylower than 0.01% by weight with respect to the amount of, respectively,safinamide and ralfinamide or the salts thereof, in particular themethanesulfonate salts.

Investigations and experimental studies carried out by the inventorshave shown that safinamide and ralfinamide and the respective salts withpharmaceutically acceptable acids prepared according to the prior artmethods contain an amount of the respective impurities (IIa) and (IIb)or the respective salts with pharmaceutically acceptable acids, such as(IIc) and (IId), that are higher than 0.03% by weight. Therefore, theabove said products are less suitable than the highly pure compounds ofthe invention. In particular, pharmaceutical preparations containingsafinamide or ralfinamide or the salt thereof with pharmaceuticallyacceptable acids, wherein the content of impurities (IIa), (IIb), andthe respective salts with pharmaceutically acceptable acids is not lowerthan 0.03%, preferably than 0.01% by weight with respect to the abovesaid active substances, are less suitable as medicaments.

In this specification and claims the values of the above indicatedlimits, unless as otherwise specified, are to be intended as expressingthe per cent ratio by weight of the “active substances”, i.e., theeffective content of the biologically active impurity (IIa, IIb) withrespect to the effective content of the therapeutically active substance(Ia, Ib).

The process described in this invention by strongly reducing theimpurities leads to products with high chemical purity and saferbiological profile. Other impurities, barely detectable, derive from thevery small quantities of 2- and 4-fluorobenzyl chloride and of 3- and4-fluorobenzyl chloride which are contained in the commerciallyavailable 3-fluorobenzyl chloride and 2-fluorobenzyl chloriderespectively, used for the synthesis of4-(3-fluorobenzyloxy)benzaldehyde (IVa) and4-(2-fluorobenzyloxy)benzaldehyde (IVb) intermediates for thepreparation of, respectively, compounds (Ia) and (Ib). According to theprocess described in the present invention safinamide and ralfinamideare obtained with high yields and high purity where the content of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]-propanamide(IIa) and(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]-propanamide(IIb), and their salt, in particular with methanesulfonic acid(generically named “dibenzyl derivatives”) in safinamide and ralfinamideor the salt thereof, in particular with methanesulfonic acid, is lowerthan or equal to 0.03%, preferably 0.01% (by weight).

The process object of the present invention starts from4-hydroxy-benzaldehyde and comprises the three following steps:

a) O-benzylation of 4-hydroxybenzaldehyde with derivatives of thefollowing general formula 3- or 2- F—C₆H₄—CH₂-Y, where Y is a leavinggroup (Cl, Br, I, OSO₂CH₃ etc.); this O-benzylation is carried out underconditions which are highly selective for O-alkylation and gives4-(3-fluorobenzyloxy)benzaldehyde and 4-(2-fluorobenzyloxy)benzaldehydeof high purity;

b) reductive alkylation of L-alaninamide, base or salt, with4-(3-fluorobenzyloxy)benzaldehyde and 4-(2-fluorobenzyloxy)benzaldehyde,where the reducing system is hydrogen gas and a heterogeneous catalyst,for obtaining, after crystallization, safinamide and ralfinamiderespectively in very high enantiomeric and chemical purity;

c) preparation of safinamide and ralfinamide salts with apharmaceutically acceptable acid by salification of safinamide andralfinamide respectively, obtained in the preceding step.Pharmaceutically acceptable acids are, for instance, selected fromnitric, hydrochloric, hydrobromic, sulphuric, perchloric, phosphoric,methanesulfonic, p-toluensulfonic, acetic, trifluoroacetic, proprionic,glycolic, lactic, oxalic, malonic, malic, maleic, tartaric, citric,benzoic, cinnamic, mandelic and salicylic acid.

A further object of this invention is to provide safinamide andralfinamide or their salts with a pharmaceutically acceptable acid,preferably methanesulfonic acid, with a high purity degree, inparticular with a content of the respective dibenzyl derivatives of theformula (IIa) or (IIb) or the salts thereof with a pharmaceutically byacceptable acid, e.g. the methanesulfonic acid, lower than 0.03%,preferably lower than 0.01% by weight (referred to the “activesubstances”), which is suitable for their use as medicaments. Moreover,another object of this invention is to provide pharmaceuticalformulations comprising safinamide or ralfinamide or a salt thereof witha pharmaceutically acceptable acid, preferably methanesulfonic acid, asthe active agents wherein the content of the respective dibenzylderivatives (IIa) and (IIb) or the salt thereof with a pharmaceuticallyacceptable acid, e.g. methanesulfonic acid, is lower than 0.03%,preferably lower than 0.01% (by weight) with respect to the above saidactive agents. These new pharmaceutical formulations were neithersuggested nor achievable by applying the pharmaco-toxicologicalknowledge regarding safinamide and ralfinamide nor by using these activeagents prepared according to the methods available in the state of theart.

Therefore, said pharmaceutical formulations comprising safinamide orralfinamide or the salts thereof with a pharmaceutically acceptableacid, preferably methanesulfonic acid, having the above said high puritydegree constitute a further object of this invention.

The above said pharmaceutical formulations may optionally comprise oneor more additional active agents, besides safinamide or ralfinamide orthe salts thereof with a pharmaceutically acceptable acid, preferablymethanesulfonic acid, having the above described high purity degree.

For instance, a new pharmaceutical formulation useful for the adjunctivetreatment of Parkinson's disease or restless legs syndrome may compriseone or more adjunctive Parkinson's disease active agent(s) such as thosedescribed in WO 04/089353 and WO 05/102300, preferably a dopamineagonist and/or levodopa and/or a catechol-O-methyltransferase (COMT)inhibitor, in addition to safinamide or a salt thereof with apharmaceutically acceptable acid, preferably methanesulfonic acid,having the above said high purity degree. As a further example, a newpharmaceutical formulation according to this invention useful for thetreatment of pain conditions, including chronic pain and neuropathicpain, and migraine may contain a further active agent such as gabapentinand pregabalin or a pharmaceutically acceptable salt thereof asdescribed in EP 1423168, in addition to ralfinamide or a salt thereofwith a pharmaceutically acceptable acid, preferably methanesulfonicacid, having the above said high purity degree.

The pharmaceutical compositions containing high purity safinamide orralfinamide according this invention can be prepared by conventionalprocedures known in the art, for instance by mixing the active compoundswith pharmaceutically, therapeutically inert organic and/or inorganiccarrier materials. The compositions of the invention can be in liquidform, e.g. in the form of a solution, suspension, emulsion; or in solidform, e.g. tablets, troches, capsules, patches.

Suitable pharmaceutically, therapeutically inert organic and/orinorganic carrier materials useful in the preparation of the compositionof the present invention include, for example, water, gelatine, arabicgum, lactose, starch, cellulose, magnesium steareate, talc, vegetableoils, polyalkyleneglycols, cyclodextrins and the like. Thepharmaceutical compositions of the invention can be sterilized and maycontain, besides the active ingredient(s), further components well knownto the skilled in the art, such as, for example, preservatives,stabilizers, wetting or emulsifying agents, e.g. paraffin oil, mannidemonooleate, salts to adjust osmotic pressure, buffers and the like. Afurther object of this invention is to provide a method for treating CNSdisorders, in particular epilepsy, Parkinson's disease, Alzheimer'sdisease and restless legs syndrome, comprising administering to apatient in need thereof an effective amount of high purity safinamide ora salt thereof with a pharmaceutically acceptable acid, preferablymethanesulfonic acid, having a content of dibenzyl derivative (IIa) or asalt thereof with a pharmaceutically acceptable acid, preferablymethanesulfonic acid, lower than 0.03%, preferably lower than 0.01% byweight (referred to the “active substances”). Said method includestreating Parkinson's disease or restless legs syndrome by administeringto a patient in need thereof an effective amount of the high puritysafinamide described above, optionally in conjunction with one or moreParkinson's disease active agent(s) as described in WO 2004/089353, suchas, for instance, a dopamine agonist and/or levodopa and/or acatechol-O-methyltransferase (COMT) inhibitor.

Moreover, a further object of this invention is to provide a method fortreating pain conditions including chronic pain and neuropathic pain,migraine, bipolar disorders, depressions, cardiovascular, inflammatory,urogenital, metabolic and gastrointestinal disorders comprisingadministering to a patient in need thereof an effective amount of highpurity ralfinamide or a salt thereof with a pharmaceutically acceptableacid, preferably methanesulfonic acid, having a content of dibenzylderivative (IIb) or a salt thereof with a pharmaceutically acceptableacid, preferably methanesulfonic acid, lower than 0.03%, preferablylower than 0.01% by weight (referred to the “active substances”).

The above said method includes treatment of pain conditions, includingchronic pain and neuropathic pain, and migraine with high purity degreeralfinamide or a salt thereof with a pharmaceutically acceptable acid,preferably methanesulfonic acid, optionally in conjunction withgabapentin or pregabalin.

In WO 90/14334, and in the paper by Pevarello et al. in J. Med. Chem.,1998, 41, 579-590, a three steps process for the preparation ofbenzyloxy-benzylamino-alkanamides is described:

a) synthesis of the intermediate 4-benzyloxybenzaldehydes byO-benzylation of the corresponding 4-hydroxybenzaldehydes with thesuitable benzyl chlorides

b) reductive alkylation of α-amino-amides with 4-benzyloxy-benzaldehydesusing sodium cyanoborohydride as a reducing agent as schematically shownhere below

where R represents, among other substituents, 3-F and 2-F; R¹represents, among other substituents, hydrogen; R² represents, amongother substituents, hydrogen; R³ represents, among other substituents,CH₃; both R⁴ and R⁵ represent, among other substituents, hydrogen.

In particular, as far as safinamide and rafinamide preparation isconcerned, the reductive alkylation is the reductive alkylation ofL-alaninamide with 4-(3-fluorobenzyloxy)benzaldehyde and4-(2-fluorobenzyloxy)benzaldehyde respectively as shown here below

In J. Med. Chem. (Pevarello et al.), 1998, 41, 579-590 yields of 45% and60% for the preparation of safinamide and ralfinamide methanesulfonaterespectively, are reported, starting from the corresponding(fluorobenzyloxy)benzaldehydes.

The process described in WO 90/14334 and in the above cited paper is thesame and provides a one-pot system where the iminoalkylation and thereduction are made in the same reactor. The suitable aldehyde is addedall at once to a mixture of L-alaninamide hydrochloride, sodiumcyanoborohydride, methanol and powdered molecular sieves.

According to Pevarello et al., in Org. Prep. Proc. Int. 1996, 28,179-183 (where the synthesis of some α-benzylaminoamide derivatives byreductive alkylation is described), use of an α-aminoamide ashydrochloride is important for the formation of the iminium ion in placeof the corresponding imine, as the iminium ion reacts more easily withsodium cyanoborohydride than with the aldehyde carbonyl group.

According to above authors, the one-pot procedure seems to avoidSchiff-base racemization problems and the molecular sieves speed up thereaction (although the yields are poor).

The cyanoborohydride is the only reducing agent utilized, and it seemsthat this choice is due to its low reactivity and its selectivity (seeReview “Sodium Cyanoborohydride—A Highly Selective Reducing Agent forOrganic Functional Groups”—C. F. Lane, Synthesis 1975, 132-146), whichmakes it able to distinguish between the protonated Schiff base and thestarting aldehyde. The synthesis described in the paper by Pevarello etal. provides the isolation of the products by column chromatography,followed by conversion into the corresponding salts by treatment withacids. No information is provided about the enantiomeric and/or chemicalpurity of both safinamide and ralfinamide and/or their salts.

The method described above suffers from many drawbacks, that limit itsuse on large scale; herebelow some examples of said drawbacks arelisted:

-   -   formation of cyanides;    -   formation of boron derivatives, difficult to remove from the        active principles;    -   use of powdered molecular sieves which are physically changeable        and expensive;    -   low yields;    -   low final product concentration in the reductive alkylation        reaction mixture (about 2-3% weight/volume);    -   isolation of the products by column chromatography, which is        considered a troublesome and expensive purification method when        large scale preparations of active agents through chemical        synthesis are involved.

Moreover, as shown in the examples which follow this description, theproducts obtained according to the methods described above contain anamount of impurities (IIa), (IIb), (IIc) or (IId) which is higher than0.03% by weight with respect to the respective active substance (Ia),(Ib), (Ic) or (Id). In addition, it has been shown that it is difficultto eliminate said impurities from the final product safinamide andralfinamide or their salts, by using commonly known purification methodssuch as crystallization from solvents or chromatography, which in anycase imply a reduction of yields.

Synthesis of the 4-(fluorobenzyloxy)-benzaldehyde Intermediates

According to the known methods, the fluorobenzyloxy-benzaldehydesintermediates for the synthesis of safinamide and ralfinamide areobtained by benzylation of 4-hydroxybenzaldehyde in a basic medium, thatis by benzylation of phenol salts which, being ambident nucleophiles,give two different products, i.e. the desired O-alkylated derivativesand the undesired C-alkylated derivatives.

It has been effectively found that the fluorobenzylation of4-hydroxybenzaldehyde with 3-fluorobenzyl chloride, performed accordingto the known methods, gives the 4-(3-fluorobenzyloxy)benzaldehyde (IVa)as the main product together with3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde (Va) that derivesfrom the alkylation of both the hydroxy group in position 4 and carbonatom in position 3 of the 4-hydroxybenzaldehyde. The same happens in thefluorobenzylation of 4-hydroxybenzaldehyde with 2-fluorobenzyl chlorideaccording to the following scheme:

The reductive alkylation of L-alaninamide with an aldehyde whichcontains the di-alkylated impurity gives a safinamide or ralfinamide endproduct which is also impure of the respective di-alkylated compound,the di-benzyl derivative, whether as a free base (IIa) or (IIb) or asalified compounds, preferably with methanesulfonic acid (IIc) or (IId),as shown in the following scheme:

Other pharmaceutically acceptable acids, e.g. nitric, hydrochloric,hydrobromic, sulphuric, perchloric, phosphoric, methanesulfonic,p-toluensulfonic, acetic, trifluoroacetic, proprionic, glycolic, lactic,oxalic, malonic, malic, maleic, tartaric, citric, benzoic, cinnamic,mandelic and salicylic acid can be used in the place of the preferredmethanesulfonic acid. The mono-alkylated derivative (safinamide orralfinamide) and the corresponding di-alkylated impurities have similarchemical-physical properties and this makes difficult the purificationof safinamide and ralfinamide with traditional methods.

Furthermore the known methods suffer from these additional drawbacks:

1) the use of a lower alcohol as a solvent; in basic conditions, thesolvent, for example methanol, can act itself as a nucleophilic reagentand gives, with 3- or 2-fluorobenzyl chloride, a certain amount ofmethyl-fluorobenzyl-ether;

2) the extraction of the final product with a water-immiscible organicsolvent is possible only after the alcoholic reaction solvent has beeneliminated from the reaction mixture.

It has now been found that by using the above methods, in order toobtain a final product of formula (Ia) or (Ib) wherein the content ofthe respective impurity (Ia) or (IIb) is lower than 0.03% (by weight),it is necessary to drastically purify the intermediate4-(3-fluorobenzyloxy)benzaldehyde (IVa) or4-(2-fluorobenzyloxy)benzaldehyde (IVb) to reduce content of therespective impurities of formula (Va) and (Vb).

Said purification is preferably carried out by submitting the reactionproducts to crystallization, more preferably by adding to a solution ofthe crude compound (IVa) or (IVb) in an inert organic solvent a miscibleinert organic non-solvent. The organic inert solvent is preferablyselected from the aromatic hydrocarbons and, more preferably, istoluene. The miscible inert organic non-solvent is preferably selectedfrom the lower aliphatic hydrocarbons, more preferably is n-hexane. Afurther crystallization procedure may consist in dissolving the abovesaid compounds (IVa) or (IVb) into a hot solvent, e.g. cyclohexane or adi(C₃-C₄)alkyl ether, such as diisopropyl ether at reflux, and thencooling the solution at room temperature, preferably at 10-15° C., mostpreferably, with inducing crystallization by addition of pure crystalsof the pure compound (IVa) or (IVb).

According to one aspect of this invention, it has now surprisingly beenfound that when the reaction between an alkylating agent of formula(IIIa) or (IIIb) (see the scheme below where the F atom is in position 2or 3 and Y is a leaving group such as, for example, Cl, Br, I, OSO₂CH₃,OSO₂C₆H₄-pCH₃, etc.) and 4-hydroxybenzaldehyde, is carried out underphase-transfer conditions, the corresponding4-(fluorobenzyloxy)benzaldehydes are obtained in high yields and withvery low level of C,O-bis-alkylated impurities.

This new fluorobenzylation of 4-hydroxybenzaldehyde under phase-transferconditions can be made both in a solid/liquid system, where in theliquid organic phase the reagents and the phase-transfer catalyst aredissolved and the solid phase is constituted by the inorganic base orthe 4-hydroxybenzaldehyde salt (possibly generated in situ from4-hydroxy-benzaldehyde and the inorganic base itself), and in aliquid/liquid organic/aqueous system where the inorganic base isdissolved in the aqueous phase.

A preferred system is the solid/liquid system wherein the inorganic baseis preferably selected from Na₂CO₃, K₂CO₃, KOH, NaOH.

The organic solvents used in the reaction, both in the case of theliquid/liquid system and of the solid/liquid system, can be dialkylethers such as, for example, di-tert-butyl ether, ethyl-tert-butylether, or aromatic hydrocarbons such as, for example, toluene,ethylbenzene, isopropylbenzene and xylenes. All these solvents can beeasily recovered by distillation.

The phase-transfer catalysts employed can be quaternary ammonium orphosphonium salts such as, for example, tetrabutyl ammonium bromide,tetradecyltrimethyl ammonium bromide, hexadecyltributyl phosphoniumbromide, tricaprilylmethyl ammonium chloride (Aliquat), methyltrialkyl(C₈-C₁₀)ammonium chloride (Adogen), the tetradecyltrimethyl ammoniumbromide being the preferred one.

Also polyethyleneglycols of low molecular weight can be used asphase-transfer catalysts such as, for example, PEG-200 (CAS 25322-68-3)or PEG-400 (CAS 25322-68-3).

The quantity of phase-transfer catalyst used is between 0.02-1 mol permole of 4-hydroxybenzaldehyde, preferably between 0.1-1 mol per mole of4-hydroxybenzaldehyde as, in these conditions, the quantity of theC,O-bis-fluorobenzylated impurities may result to be less than 0.03%,preferably equal to 0.01% or less by weight.

The ratio between the alkylating agents of formula (IIIa) or (IIIb) and4-hydroxybenzaldehyde is between 0.6 and 1.5, preferably between 0.9 and1.1.

The reaction temperature is between 60° C. and 160° C., the preferredinterval being between 80° C. and 120° C.

The reaction time is generally between 4 and 8 hours.

The reaction yields are very high, as a rule more than 90%.

The reaction productivity, i.e. the concentration of the reactionproducts in the reaction mixture is very high in the reaction conditiondescribed, normally is more or equal to 25% (weight/volume).

Synthesis of safinamide and ralfinamide by reductive alkylation of α-aminoamides

The state of the art would suggest to the expert in the field that thereductive alkylation of α-amino-amides with 4-(3- or2-fluorobenzyloxy)benzaldehydes using hydrogen and a heterogeneouscatalyst as a reducing agent, should not be suitable for the preparationof safinamide and ralfinamide because of the incompatibility among thereagents and the final products and the reduction conditions.

In fact, it is well known how easily benzaldehydes are reduced to benzylalcohols or even to the corresponding hydrocarbons, as well as it isknown that the conditions which should be used to perform a reductivealkylation with hydrogen and a heterogeneous catalyst, are normally thesame conditions used to break the bonds between a benzylic carbon atomand heteroatoms like nitrogen or oxygen, the kind of bonds that arepresent both in safinamide and ralfinamide and in their precursors.

In fact, the benzylic group is normally employed as a protecting groupof phenols or amines (see “Protective Groups in Organic Synthesis”, T.W. Greene and P. G. M. Wuts, 3^(rd) Edition, 1999, John Wiley & Sons,Inc.) because of the easiness of its introduction and successive removalby catalytic reduction. In the reductive alkylation for obtainingsafinamide and ralfinamide, one could expect the formation of manyby-products, some of which are reported here below:

The fact that safinamide and ralfinamide have been obtained in very highyields and purity by reductive alkylation of L-alaninamide, with 4-(3-or 2-fluorobenzyloxy)benzaldehyde, using hydrogen and a heterogeneouscatalyst as a reducing system, is a surprising and innovative aspect ofthis synthetic procedure.

Moreover, the reaction conditions which are used according to thisprocedure are easily applicable to the production in bulk.

The reductive alkylation, object of the present invention, is performedin two steps:

a) formation of the Schiff base

b) catalytic reduction of the Schiff base

The two steps can be performed in succession in the same reactor (onepot reaction) either with, or without, isolation of the Schiff base, inboth cases with high yields.

In the case of isolation of the Schiff base, the experimental conditionsapplied for its formation allow to obtain the isolated Schiff base inthe form of a precipitate in high yields and very pure form.

The Schiff base preparation is suitably performed in an organic proticsolvent, that must be inert vs. the reagents and the products and alsoinert vs. the reduction conditions of the iminic double bond, such asfor example, a (C₁-C₅) lower alkanol, preferably methanol, ethanol andisopropanol.

The formation of Schiff base must be complete and this is a relevantfactor for having high yields in the subsequent catalytic reductionstep. It is therefore preferable to isolate the Schiff bases (VIa) and(VIb) before performing the reduction of the iminic double bond.

The isolated imino compounds (VIa) and (VIb) are useful intermediatesfor the preparation of safinamide and ralfinamide, respectively,according to this invention.

Alternatively one can favour the iminoalkylation reaction completion, byoperating under such conditions as to cause the precipitation of theimino compounds (VIa) and (VIb) and to submit to the catalytic reductionthe suspension containing the intermediate imino derivative.

The ratio between L-alaninamide (base or salt) and 4-(3- or2-fluorobenzyloxy)benzaldehyde can be 1:1 but also a 10% excess ofL-alaninamide can be advantageously used.

The L-alaninamide may be introduced either as a free base or as an acidaddition salt thereof. Preferably, it is introduced in the reactionmixture as a salt, most preferably as hydrochloride salt, together withthe stoichiometric amount of a base, preferably a tertiary amine suchas, for example triethylamine or diisopropylethylamine.

The reaction temperature in the preparation of the Schiff base iscomprised between 0° C. and 60° C., preferably between 20° C. and 30° C.

The reaction times are between 1 hour and 15 hours, preferably between 4hours and 6 hours.

The reduction of the Schiff base with hydrogen and a heterogeneouscatalyst is started only when the Schiff's base formation is completed:if it is started before, secondary reactions become important, sometimesprevalent, with loss in yields and purity. One of these secondaryreactions, the more important, causes the formation of benzylic alcoholsby reduction of the carbonyl group of the (fluorobenzyloxy)benzaldehydeof choice.

The preferred heterogeneous catalysts are nickel, rhodium, palladium orplatinum catalysts, on an inert support such as, for example, carbon,alumina and silica, preferably carbon and alumina, and are used in aquantity comprised between 2% and 20% of the 4-(3-or2-fluorobenzyloxy)benzaldehyde, preferably between 5% and 10%.

Platinum and palladium catalysts are the most preferred.

Platinum on active carbon, in particular, gives excellent results bothin terms of yields, which are nearly quantitative, and selectivity, asonly the iminic double bond is reduced while the bond between thebenzylic carbon atom and the heteroatoms remain unchanged. It was foundthat safinamide and ralfinamide are surprisingly stable in the reductionreaction conditions and this is an important element in industrialproduction of large quantities of safinamide or ralfinamide, as anincidental reaction time prolongation wouldn't damage the finalproducts.

The best results were obtained with wet 5% Pt/C (50% H₂O) and inparticular with 5% Pt on carbon powder by Engelhard S.r.l., Rome, Italy.

The hydrogenation reaction is normally performed under a hydrogenpressure comprised between 1 bar and 10 bars, preferably between 3 barsand 6 bars and at temperature comprised between 10° C. and 70° C.,preferably between 25° C. and 40° C.

The reduction times can vary from 1 hour to 20 hours, according totemperature, pressure, concentration, turbulence, etc., all factors wellknown to those skilled in the art.

The best results were obtained with reaction times of 4-6 hours.

At the end of the reaction the catalyst is recovered by filtration andreutilized or regenerated: the reaction solvent is distilled underreduced pressure, the residue is dissolved in water-immiscible organicsolvent and the inorganic salts are removed by washing with water.

The final raw safinamide or ralfinamide are recovered by removing bydistillation the organic solvent wherein they are dissolved.

The raw safinamide or ralfinamide are then purified by crystallization.The crystallization is preferably carried out by adding to a solution ofthe respective crude compound of formula (Ia) or (Ib) in an inertorganic solvent a miscible inert organic non-solvent. The organic inertsolvent is preferably selected from aromatic hydrocarbons such asbenzene, toluene, dimethyl benzene and ethylbenzene and lower alkylacetates and, more preferably, is ethyl acetate. The miscible inertorganic non-solvent is preferably selected from the lower aliphatichydrocarbons, such as hexane and heptane, and cyclohexane, morepreferably is n-hexane

The bases, are then transformed into the desired salts according toknown methods, in particular they are transformed into methanesulfonatesalt, which has the physical/chemical properties (stability,granulometry, flowability etc.) suitable for the subsequent formulationinto a pharmaceutical preparation for use as medicament.

EXAMPLE 1 Preparation of Purified 4-(2-fluorobenzyloxy)benzaldehyde(IVb) by Phase Transfer Catalysis

A mixture of 2-fluorobenzyl chloride (5 kg, 34.58 mol),4-hydroxy-benzaldehyde (3.9 kg, 31.94 mol), potassium carbonate (4.3 kg,31.11 mol) and tetradecyl trimethylammonium bromide (0.41 kg, 1.22 mol)in toluene (9.5 kg) is slowy brought, under stirring and under nitrogen,to reflux temperature and refluxed for 6 h.

The solution is then concentrated at room pressure, 3 kg of toluene areadded and distilled off and this procedure is repeated once again.

The heterogeneous mixture is then cooled to room temperature and thesolid is eliminated by filtration. The residual solvent is theneliminated under reduced pressure and to the oily residue 1.2 kg oftoluene are added. The mixture is heated to about 40° C. and seeded witha few grams of pure 4-(2-fluorobenzyloxy)benzaldehyde.

The heterogeneous mixture is stirred for 15 minutes at 35-40° C. andthen additioned of n-hexane (9 kg) at this temperature, in 30 minutes.After cooling to 0-5° C. and stirring for a further hour at thistemperature the solid is collected by filtration and dried under reducedpressure to give 6.5 kg (87.6% yield) of4-(2-fluorobenzyloxy)benzaldehyde; m.p. 56.7° C. (DSC, 5° C./min).

The above reaction is repeated on a 1:100 scale by using 39 g (0.319mol) of 4-hydroxybenzaldehyde as the starting material and following theabove described procedure, with the exception that, after elimination ofthe reaction solvent and addition of toluene to the oily residue, theobtained mixture is heated to about 30-35 (instead of 40° C.) and, afterseeding with a small amount of pure 4-(2-fluorobenzyloxy)benzaldehyde,the heterogeneous mixture is stirred for 15 minutes at 30° C. (insteadof 35-40° C.) before adding n-hexane. The yield is 66.8 g (90%) of4-(2-fluorobenzyloxy)benzaldehyde, m.p. 56.7° C. (DSC, 5° C./min),having a GC purity of 92.2 (area %, see Example 16A) and a3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde content 0.01% byweight determined by GC (see Example 16B)

(*) The yields reported in this and in the following Examples, when nootherwise specified, are intended as molar yields.

1.1 Further Purification of 4-(2-fluorobenzyloxy)benzaldehyde byCrystallization

One kilogram of the product prepared according to the proceduredescribed in Example 1 is dissolved in 2 kg of diisopropryl ether atreflux under stirring.

The solution is cooled to 50-55° C. in 10-15 minutes and seeded with afew grams of pure 4-(2-fluorobenzyloxy)benzaldehyde.

The suspension is cooled to 10-15° C. during 45-60 minutes and stirredfor an additional hour.

The precipitate is finally collected by filtration, washed with cooldiisopropyl ether (0.2 Kg) and dried under reduced pressure to give 0.93kg of 4-(2-fluorobenzyloxy)benzaldehyde with GC purity of 99.8 (area %,see Example 16A) and a content of3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde (Vb) of 0.005% byweight determined by GC according to Example 16B.

1.2 Preparation of 4-(2-fluorobenzyloxy)benzaldehyde (IVb) by PhaseTransfer Catalysis (PTC) Using Different Catalysts

4-(2-Fluorobenzyloxy)benzaldehyde is prepared by alkylation of4-hydroxybenzaldehyde (0.39 g) with 2-fluorobenzyl chloride by followingthe same procedure of Example 1, but using three different phasetransfer catalysts.

The results are reported in the following Table 4

TABLE 4 Phase Transfer Catalyst % % Experiment PCT Vb ** Yield 1.2 (a)Tetrabutyl 0.03 85.0 fosphonium bromide 1.2 (b) Aliquat 336* 0.03 88.81.2 (c) PEG 400 0.14 96.0 *Aliquat 336: tricaprylylmethylammoniumchloride ** % Vb: content of3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde (% by weight) Thecontent of Vb is determined by GC according to Example 16B.

1.3 Preparation of 4-(2-fluorobenzyloxy)benzaldehyde (IVb) by PhaseTransfer Catalysis (PTC) in Xylene

4-(2-Fluorobenzyloxy)benzaldehyde is prepared in 86.6% yield with acontent of 3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde of 0.02%by weight determined by GC (see Example 16B)by reacting4-hydroxybenzaldehyde (0.39 g) with 2-fluorobenzyl chloride according tothe same procedure of Example 1, but replacing toluene with xylene asthe solvent.

1.4 Preparation of 4-(2-fluorobenzyloxy)benzaldehyde (IVb) by PhaseTransfer Catalysis using Potassium Hydroxyde as a Base

4-(2-Fluorobenzyloxy)benzaldehyde is prepared in 86.7% yield with acontent of 3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde of 0.51%by weight determined by GC (see Example 16B) by reacting4-hydroxybenzaldehyde (0.39 g) with 2-fluorobenzyl chloride, accordingto the same procedure of Example 1, but using potassium hydroxyde (0.35mol) instead of potassium carbonate.

This product may be further purified by crystallization according toExample 1.1.

1.5 Preparation of 4-(2-fluorobenzyloxy)benzaldehyde (IVb) by PhaseTransfer Catalysis using 2-fluorobenzyl bromide

4-(2-Fluorobenzyloxy)benzaldehyde is prepared in 88.6% yield with acontent of 3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde of 0.07%by weight determined by GC (see Example 16B) by reacting4-hydroxybenzaldehyde (15.6 g) with 2-fluorobenzyl bromide instead of2-fluorobenzyl chloride according to the same procedure of Example 1.

EXAMPLE 2 Preparation of(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (ralfinamide, Ib) ofHigh Purity Degree (One Pot Reaction)

An autoclave is loaded with 4-(2-fluorobenzyloxy)benzaldehyde (2.0 kg,8.69 mol) prepared according to Example 1, and then a solution preparedapart of L-alaninamide hydrochloride (1.2 kg, 9.63 mol) andtriethylamine (0.97 kg, 9.63 mol) in methanol (9.5 kg) is added thereto.

The mixture is stirred at 20-25° C. for about 1 hour and then, afterseeding it with a few grams of(S)-2-[4-(2-fluorobenzyloxy)benzylideneamino]propanamide, the stirringis continued for additional 15 minutes. To the stirred heterogeneousmixture, methanol (1.6 kg) and wet (50% H₂O) Pt/C 5% (Engelhardcod.Escat 22, Engelhard S.r.l., Rome, Italy) (0.28 kg) are then added at20-25° C.

The air is purged from the autoclave with nitrogen and then hydrogen isintroduced at 5.0 bar and the pressure is maintained at this valueduring the hydrogenation course.

After 5 hours at 30-35° C. the reaction mixture is cooled to 15° C. and,after addition of methanol (4.8 kg) and heating to 40-45° C. thesuspension is filtered and the solid is washed with methanol (1.6 kg).

The solvent is eliminated under reduced pressure at about 30° C. and theresidue is additioned of water (5 L) at 20-25° C. on cooling and understirring, as the water addition is an exothermic process. Theheterogeneous mixture is further cooled to 15-20° C., kept at thistemperature for 1 hour and then filtered. The collected solid is washedwith cool water (4 L) and dried under reduced pressure to give 2.23 kg(85.0% yield) of ralfinamide with a HPLC purity of 98.8 (area %)determined according to the method of Example 17A and a C,O-dialkylated(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamidecontent of 0.01% by weight determined by HPLC, according to the methodof Example 17B.

2.1 Preparation of (S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide(Ib) of High Purity Degree by Using a Palladium Catalyst

A mixture of 5 g of 4-(2-fluorobenzyloxy)benzaldehyde, preparedaccording to the Ex. 1 and the corresponding amount L-alaninamidehydrochloride and triethylamine is hydrogenated according to the sameprocedure of Example 2, but using wet (50% H₂O) Pd/C 10% instead of wet(50% H₂O) Pt/C 5% to obtain (S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (Ib) in a 70% yield.

EXAMPLE 3 Preparation of(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide methanesulfonate(ralfinamide methanesulfonate, Id) of High Purity Degree

Ralfinamide (2.8 kg, 9.26 mol), prepared as described in Example 2, isdissolved in iso-propanol (19.5 kg) and kept at 65-70° C. and understirring under inert atmosphere.

After treatment with charcoal (150 g) and filtration, the solution isseeded with pure ralfinamide methanesulfonate and, methanesulfonic acid(900 g, 9.36 mol) is added in 30 minutes, under stirring and at atemperature of 50-55° C. The suspension is then cooled to 15-20° C. in 2hours and the stirring is continued for an additional hour. The solid isfinally collected by filtration and dried under reduced pressure to give3.59 kg (97.3% yield) of ralfinamide methanesulfonate.

The HPLC purity of the obtained product is 99.8 (area %, see Example17A)and the content of C,O-dialkylated(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate is 0.005% by weight (see Example 17B); m.p. 240.6° C.by DSC (5° C./min).

The enantiomeric purity of ralfinamide methanesulfonate determined witha chiral HPLC column is higher than 99.8 (area %, see Example 18).

EXAMPLE 4 Preparation of purified 4-(3-fluorobenzyloxy)benzaldehyde(IVa) in Ethanol Solution

To a mixture of 4-hydroxybenzaldehyde (1.52 kg, 12.45 mol), potassiumcarbonate, (1.72 kg, 12.45 mol), potassium iodide (0.2 kg, 1.20 mol) inethanol (13.0 kg), 1.8 kg, of 3-fluorobenzyl chloride (1.80 kg, 12.45mol) are added under stirring, at room temperature.

The mixture is gradually heated to reflux and then kept at thattemperature for 6 hours.

The reaction mixture is then allowed to cool to 25° C., the suspensionis filtered and the solid is washed with ethanol (1.0 kg); the ethanolsolutions are combined and then concentrated at reduced pressure until aresidue of approximately 3.5 kg is obtained.

To this residue, toluene (5.0 kg) and water (1.7 kg) are added, thesolvent mixture is stirred vigorously for 30 minutes and, afterseparation of the aqueous phase, the organic layer is evaporated todryness under reduced pressure to provide crude4-(3-fluorobenzyloxy)benzaldehyde.

To this product dissolved in 2 kg of toluene a seed of4-(3-fluorobenzyloxy)benzaldehyde is added under stirring at 20-25° C.,then n-hexane (3.8 kg) is added in 30 minutes and the mixture is cooledto 0° C. under stirring.

After 2 hours the solid is filtered and washed with n-hexane (1.3 kg).After drying, 2.6 kg (90.7% yield) of the desired product are obtained,with a gas-cromathographic purity of 99.9 (area %, see Example 16A) anda 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde content of 0.005%by weight determined by G.C. (area %, see Example 16B); m.p. 43.1° C. byDSC 5° C./min.

EXAMPLE 5 Preparation of 4-(3-fluorobenzyloxy)benzaldehyde (IVa) byPhase Transfer Catalysis

A mixture of 3-fluorobenzyl chloride (5 kg, 34.58 mol),4-hydroxy-benzaldehyde (3.9 kg, 31.94 mol), potassium carbonate (4.3 kg,31.11 mol) and tetradecyl trimethylammonium bromide (0.41 kg, 1.22 mol)in toluene (13.5 kg) is slowly brought to reflux temperature understirring and under nitrogen atmosphere, and then refluxed for 6 hours.

The solution is concentrated at room pressure and then 3 kg of tolueneare added and distilled off. This procedure is repeated once again.

The heterogeneous mixture is then cooled to room temperature and thesolid is eliminated by filtration. The residual solvent is eliminatedunder reduced pressure and then 1.2 kg of toluene are added to the oilyresidue. The mixture is stirred at 20-25° C. and seeded with a few gramsof pure 4-(3-fluorobenzyloxy) benzaldehyde, and then additioned ofn-hexane (9 kg) at this temperature, in 30 minutes.

After cooling to 0-5° C. and stirring for a further hour at thistemperature the solid is collected by filtration and dried under reducedpressure to give 6.5 kg (85% yield, GC purity 99.9 (area %, see Example16A) and a 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde contentof 0.008% by weight (see Example 16B).

5.1 Further Purification of 4-(3-fluorobenzyloxy)benzaldehyde (IVa) byCrystallization

One kilogram of 4-(3-fluorobenzyloxy)benzaldehyde, prepared according toExample 5, is dissolved in 2 kg of diisopropyl ether at reflux understirring.

The solution is cooled to 50-55° C. in 10-15 minutes and seeded with afew grams of pure 4-(3-fluorobenzyloxy)benzaldehyde.

The suspension is cooled to 10-15° C. during 45-60 minutes and stirredfor an additional hour.

The precipitate is finally collected by filtration, washed with cooldiisopropyl ether (0.2 kg) and dried under reduced pressure to give 0.95kg of 4-(3-fluorobenzyloxy)benzaldehyde with GC purity of 99.9 (area %,see Example 16A) and a content of3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde lower than 0.005%by weight determined by GC (see Example 16B).

5.2 Preparation of 4-(3-fluorobenzyloxy)benzaldehyde (IVa) by PhaseTransfer Catalysis using 3-fluorobenzyl bromide

4-(3-Fluorobenzyloxy)benzaldehyde is prepared in 86.1% yield with acontent of 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde of 0.07%by weight determined by GC (see Example 16B) by reacting4-hydroxybenzaldehyde (15.6 g) with 3-fluorobenzyl bromide according tothe same procedure of Example 5 but using 3-fluorobenzyl bromide insteadof 3-fluorobenzyl chloride.

The so obtained 4-(3-fluorobenzyloxy)benzaldehyde is purified accordingto Example 5.1 to yield the title product in 97.3% yield with a contentof 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde of 0.07% byweight determined by GC (see Example 16B).

5.3 Preparation of 4-(3-fluorobenzyloxy)benzaldehyde (IVa) by PhaseTransfer Catalysis using 3-fluorobenzyl methanesulfonate

4-(3-Fluorobenzyloxy)benzaldehyde is prepared in 97.5% yield with acontent of 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde of 0.45%by weight, determined by GC (see Example 16B), by reacting4-hydroxybenzaldehyde (15.6 g) with 3-fluorobenzyl methanesulfonateinstead of 3-fluorobenzyl chloride according to the same procedure ofExample 5. This product is further purified according to the procedureof the Example 5.1.

EXAMPLE 6 Preparation of(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (safinamide, Ia) ofHigh Purity Degree (One Pot Reaction)

An autoclave is loaded with, 4-(3-fluorobenzyloxy) benzaldehyde (2.0 kg,8.69 mol) prepared as in Example 4, and then a solution, prepared apart,of L-alaninamide hydrochloride (1.2 kg, 9.63 mol) and triethylamine(0.97 kg, 9.63 mol) in methanol (7.1 kg) is added thereto.

The mixture is stirred at 20-25° C. for 1 hour and, after seeding withfew grams of (S)-2-[4-(3-fluorobenzyloxy)benzylideneamino]propanamide,stirring is continued for additional 15 minutes. To the stirredheterogeneous mixture, methanol (1.8 kg) and, wet (50% H₂O) Pt/C 5%(Engelhard cod. Escat 22)(0.3 kg) are then added, at 20-25° C.

The air is purged from the autoclave with nitrogen and then hydrogen isintroduced at 5.0 bars.

After 5 hours at 30-35° C., the mixture is cooled to 15° C., methanol(4.8 kg) is added and the mixture is heated to 40-45° C.; finally thesolid is filtered out and washed with methanol (1.6 kg).

The solvent is eliminated under reduced pressure approximately at 30° C.and then a mixture of ethyl acetate (23.0 kg) and water (18.0 kg) isadded to the residue. After stirring for 15 minutes, the aqueous phaseis separated and extracted with ethyl acetate (7.0 kg). The collectedorganic phases are concentrated until a residue of approximately 6.0 kgis obtained. To this residue n-heptane (10.8 kg) is added and themixture is stirred at 20° C. for about 2 hours. The solid is thencollected by filtration and washed with n-heptane.

After drying the solid under reduced pressure, 2.41 kg (91.8% yield) ofthe title compound are obtained with a HPLC purity of 98.4 (area %, seeExample 17A), and a content of C,O-dibenzylated(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamide)of 0.005% by weight (see Example 17B).

6.1 Preparation of (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide(Ia) of High Purity Degree by Using a Pd Catalyst

(S)-2-[4-(3-Fluorobenzyloxy)benzaldehyde (5 g) in the presence of thecorresponding amounts of L-alaninamide hydrochloride and triethylamineis hydrogenated according to the same procedure of Example 6, by usingwet (50% H₂O) Pd/C 10%, instead of wet (50% H₂O) Pt/C 5%, to produce Iain a 72% yield.

6.2 Preparation of (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide(Ia) of High Purity Degree by hydrogenation at 1 bar

A mixture of (S)-2-[4-(3-fluorobenzyloxy)benzaldehyde, L-alaninamidehydrochloride and triethylamine is hydrogenated according to the sameprocedure of Example 6, but at 1 bar/H₂ instead of 5 bar/H₂. The yieldof (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide is 90% with aHPLC purity of 98.7 (area %, see Example 17A) and a content of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamideof 0.005% by weight determined by HPLC (see Example 17B).

6.3 Preparation of (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide(Ia) of High Purity Degree (One Pot Reaction) by using L-alaninamidebase

(S)-2-[4-(3-fluorobenzyloxy]benzaldehyde (10 g) is reacted according tothe same procedure of Example 6, but using L-alaninamide base, insteadof its hydrochloride and triethylamine. The yield of(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide is 92% with a HPLCpurity of 99.7 (area %, see Example 17A) and a content of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamidelower than 0.005% by weight determined by HPLC (see Example 17B).

EXAMPLE 7 Preparation of(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide methanesulfonate(safinamide methanesulfonate, Ic) of High Purity Degree

Safinamide (2.41 kg, 7.97 mol), prepared as described in Example 6, isdissolved in ethyl acetate (56.5 kg) at 65° C. and decoloured withcharcoal (100 g).

After filtration, the solution is stirred and seeded with a few grams ofsafinamide methanesulfonate and, after 15 minutes, methanesulfonic acid(850 g, 8.84 mol) is added in 30 minutes, at a temperature of 50-55° C.The suspension is cooled under stirring to 20-25° C. during 2 hours andstirred for an additional hour. The precipitate is finally collected byfiltration and dried under reduced pressure to give 2.83 kg (89.1%yield) of safinamide methanesulfonate.

The content of impurity(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate (IIc) measured by HPLC (see Example 17B) is of 0.005%by weight. The title compound has m.p. 216.8° C. by DSC (5° C./min).

The enantiomeric purity, measured with a chiral HPLC column, is over99.9 (area %, see Example 19).

¹H-NMR (D₂O) (Bruker A V300) δ (ppm, with respect to H₂O at 4.7 ppm):1.43 (3H, d, J=7 Hz, CH₃); 2.66 (3H, s, CH₃SO₃H); 3.87 (1H, q, J=7 Hz,H-2); 3.97 (2H, bs, CH₂NR); 4.89 (2H, s, CH₂OR); 6.88 and 7.23 (4H,AA′XX′ aromatic p-disubstituted system,; 6.90÷7.22 (4H, aromatic H)

¹³C-NMR (D₂O) (Bruker AV300) δ ppm: 15.68 (CH₃); 38.27 (CH₃SO₃H); 48.99(CH2NR); 54.81 (CH); 69.00 (OCH₂); 114.15 (d, J_(C-F)=21 Hz, aromaticCH); 114.76 (d, J_(C-F)=20 Hz, aromatic CH); 115.38 (aromatic CH);123.06 (d, J_(C-F)=24 Hz, aromatic CH); 123.24; 130.29 (d, J_(C-F)=6 Hz,aromatic CH); 131.54 (aromatic CH); 138.76 (d, J_(C-F)=7 Hz, aromaticCH); 158.52; 162.89 (d, J_(C-F)=245 Hz, C-F); 171.92 (CO)

EXAMPLE 8 Preparation of(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (safinamide, Ia) ofHigh Purity Degree, with Isolation of the Intermediate Schiff base(S)-2-[4-(3-fluorobenzyloxy)benzylideneamino]propanamide (VIa) a)(S)-2-[4-(3-Fluorobenzyloxy)benzylideneamino]propanamide (VIa)

To a suspension of 4-(3-fluorobenzyloxy)benzaldehyde (60.0 g 0.26 mol),prepared as in the Example 5 and L-alaninamide hydrochloride (35.7 g0.29 mol) in methanol (280 mL), triethylamine (29.1 g, 0.29 mol) isadded at room temperature with stirring under nitrogen atmosphere.Stirring is maintained for one additional hour.

The solution is then seeded with a few mg of(S)-2-[4-(3-fluorobenzyloxy)benzylideneamino]propanamide, thetemperature is lowered to 5-10° C. and the stirring continued for 2hours.

The solid is collected by filtration and washed with methanol at 0° C.

After drying it at reduced pressure, 57.3 g (73.2% yield) of the titlecompound is obtained with m.p. 112.0° C. by DSC (5° C./min).

¹H-NMR (DMSO-d₆) (Bruker AV300) δ (ppm, with respect to TMS at 2.55 ppm;DMSO solvent at 3.35 ppm): 1.31 (3H, d, J=7 Hz, CH₃); 3.86 (1H, q, J=7Hz, H-2); 5.18 (2H, s, CH₂OR); 7.08 and 7.79 (4H, AA′XX′ p-disubstitutedaromatic system); 7.10-7.50 (4H, m, aromatic H); 8.27 (1H, s, CH═NR).

¹³C-NMR (DMSO-d₆) (Bruken AV300) δ (ppm): 20.5 (CH3); 67.6 (CH); 68.4(OCH2); 114.1 e 114.4 (d, J_(C-F)=21 Hz, aromatic)CH;114.5 e 114.8 (d,J_(C-F)=21 Hz; aromatic CH; 114.8 (aromatic CH); 123.5 (d, J_(C-F)2 Hz,aromatic CH); 129.0 and 129.9 (aromatic CH); 130.4 and 130.5 (d,J_(CF)=7 Hz, aromatic CH); 139.6 and 139.7 (d, J_(C-F)=6 Hz aromaticquaternary C); 160.2; 160.5 and 163.8 (d, J_(C-F)=245 Hz C-F); 160.6(CH═N);174.8 (CO)

b) (S)-2-[4-(3-Fluorobenzyloxy)benzylamino]propanamide (Ia)

An autoclave is loaded with(S)-2-[4-(3-fluorobenzyloxy)benzylidene-amino]propanamide (16.0 g; 0.053mol), prepared as described above, and wet (50% H₂O) Pt/C 5% (1.7 kg:Engelhard S.r.l., Rome, Italy) and methanol (90 mL) is added thereto.The autoclave is purged from air with nitrogen and then hydrogen isintroduced at 5.0 bars. The reaction is kept at 5.0 bar and at 35° C.for 1 hour. After cooling to room temperature and elimination of thecatalyst by filtration, the solvent is distilled off under reducedpressure until a residue of approximately 30 g is obtained. To thisresidue a mixture of ethyl acetate (150 mL) and H₂O (110 mL) is addedand the heterogeneous mixture is heated to 40° C., until two clearphases are obtained. These two phases are separated and the aqueouslayer is extracted with 50 mL of ethyl acetate at 40° C. The organicphases are collected and evaporated to dryness. The procedure isrepeated twice by adding every time 90 mL of ethyl acetate to make theproduct anhydrous. 95 mL of n-heptane are then added slowly and understirring to the residue. The mixture is then maintained under stirringfor 3 hours at 20° C. The solid formed is collected by filtration,washed with n-hetpane (15 mL) and dried under reduced pressure to give15.2 g (94.8% yield) of safinamide with a HPLC purity of 99.8 (area %,see Example 17A)and a content of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidelower than 0.005% by weight measured by HPLC (see Example 17B).

EXAMPLE 9 Preparation of(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (ralfinamide, Ib) ofHigh Purity Degree, with Isolation of the Intermediate Schiff Base(S)-244-(2-fluorobenzyloxy)benzylideneaminol propanamide (VIb) a)(S)-2-[4-(2-Fluorobenzyloxy)benzylideneamino]propanamide (VIb)

(S)-2-[4-(2-Fluorobenzyloxy)benzylideneamino]propanamide is prepared ina 88% yield, m.p. 121° C. (capillary), by following the same procedureof Example 8, step a) but using 4-(2-fluorobenzyloxy)benzaldehydeinstead of 4-(3-fluorobenzyloxy)benzaldehyde.

¹H-NMR: (CDCl₃, 300 MHz, 298K) δ (ppm, with respect to TMS): 1.46 (3H,d, J=7.0 Hz, CH₃); 3.91 (1H, q, J=7.0 Hz, CH—CO); 5.17 (2H, s, O—CH₂);7.02 (2H, d, J=8.9 Hz aromatic H ortho to O—CH₂); 7.09 (1H, ddd,J_(H-F)=9.78 Hz J_(orto)=8.55 Hz J_(meta)=1.23 Hz aromatic H ortho toF); 7.15 (1H, dt, J_(orto)=7.35 Hz J_(meta)=1.23 Hz aromatic H para toF); 7.27-7.40 (1H, m, aromatic H para to CH₂); 7.48 (1H, dt,J_(orto)=J_(H-F)=7.35 Hz J_(meta)=1.53 Hz aromatic H ortho to CH₂); 7.71(2H, d, J=8.9 Hz aromatic H ortho to CH═N); 8.17 (1H, s, C═N)

¹³C-NMR: (CDCl₃, 75.4 MHz, 298K) δ (ppm): 21.4 (CH₃); 63.8 (OCH₂); 68.4(H₂NCOCH); 115.0 (d, J_(C-F)=22.4 Hz, aromatic CH), 115.5 (d,J_(C-F)=20.7 Hz, aromatic CH); 123.7 (d, J_(C-F)=14.4 Hz, quaternaryaromatic C); 124.5 (bd, aromatic CH); 129.0 (quaternary aromatic C);129.8 (bd, aromatic CH); 130.1 (bd, 2 aromatic CH); 160.5 (d,J_(C-F)=246.4 Hz, quaternary aromatic C); 161.1 (aromatic C-0); 161.1(C═N); 176.9 (CONH₂)

b) (S)-2-[4-(2-Fluorobenzyloxy)benzylamino]propanamide (Ib)

(S)-2-[4-(2-Fluorobenzyloxy)benzylamino]propanamide is prepared in a 93%yield from (S)-2-[4-(2-fluorobenzyloxy)benzylideneamino]propanamide byfollowing the same procedure of Example 8, step b). The content of(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideis 0.02% by weight determined by HPLC (see Example 17B).

EXAMPLE 10 Preparation of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate (IIc) a)3-(3-Fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde (Va)

In a 4 L round bottomed flask kept under nitrogen atmosphere,4-hydroxy-benzaldehyde (400 g, 3.28 mol), potassium carbonate (453 g,3.28 mol), toluene (2 L) and 3-fluorobenzylchloride (1400 g, 9.68 mol)are added in sequence and the mixture is refluxed under stirring for 5days. At this point a GC analysis reveals that the reaction mixturecontains 4-(3-fluorobenzyloxy)benzaldehyde and3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde in a ratio of91.4:8.6 (area/area, see Example 16A).

The reaction mixture is cooled to room temperature and then 2 L of waterare added under stirring. The organic phase is separated and the solventis distilled under reduced pressure (20 mmHg) at 35° C. until no moresolvent passes over. The pressure is then lowered to 3 mmHg and theexternal temperature is raised up to 300° C. and the fraction thatdistils between 255° C. and 265° C., (40.6 g), is collected.

A GC analysis shows a area/area ratio of C,O-dibenzylated derivative(Va) of the title compound vs. the monoalkylated one (IVa) of 99.6:0.4.(area/area, see Example 16A).

¹H-NMR (CDCl₃) (Bruker AV300) δ (ppm, with respect to TMS): 4.05 (2H, s,CH₂); 5.13 (2H, s, OCH₂); 6.85-7.40 (9H, m, aromatic H); 7.73-7.79 (2H,m, aromatic H ortho to C═O); 9.88 (s, CHO).

¹³C-NMR (CDCl₃) (Bruker AV300) δ (ppm): 36.1 (CH2); 69.4 (CH2O); 111.4(aromatic CH);112.9 and 113.2 (d, J_(C-F)=20 Hz, aromatic CH), 113.9 and114.2 (d, J_(C-F)=22 Hz, aromatic CH); 114.9 and 115.0 (d, J_(C-F)=21Hz, aromatic CH ; 115.7 e 115.9 (d, J_(C-F)=25 Hz aromatic CH); 122.6(d, J_(C-F)=3 Hz, aromatic CH); 124.4 (d, J_(C-F)=3 Hz, aromatic CH);129.6 and 129.8 (d, J_(C-F)=8 Hz, aromatic CH); (d, J_(C-F)=7 Hz,quaternary aromatic C); 129.9 (C quaternary aromatic C); 130.0(quaternary aromatic C); 130.1 and 130.2 (d, J_(C-F)7Hz, CH aromatic);131.2 (aromatic CH); 131.5 (aromatic CH); 138.3 (d, J_(C-F)=7 Hz,quaternary aromatic C); 142.3 (d, J_(C-F)=7 Hz, quaternary aromatic C);161.0, 161.2 and 164.4 (d, J_(C-F)=240, 2 C-F overlapping); 190.8 (CHO).

b)(S)-2-[3-(3-Fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamide(IIa)

To 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde (35.6 g, 0.105mol) in a 500 mL flask, a solution previously prepared by cautiouslyadding under stirring triethylamine (12 g, 0.119 mol) to a 170 mLmethanol solution of L-alaninamide hydrochloride (14.8 g, 0.119 mol), isadded at room temperature. This reaction mixture is stirred for 1 hourat room temperature and then it is transferred to a 1.8 L autoclave andadditioned of 3.4 g of wet (50% H₂O) Pt/C 5%.

The air is purged from the autoclave with nitrogen and then hydrogen isintroduced at 5.0 bar.

The reaction is performed at a temperature of 35° C. for 3-5 hours.

After cooling to room temperature and eliminating the catalyst byfiltration, the solvent is distilled off under reduced pressure until aresidue of approximately 65 g is obtained. To this residue a mixture ofethylacetate (340 mL) and water (250 mL) is added and the heterogeneousmixture is warmed to 40° C. and kept at this temperature withoutstirring, until two clear phases are obtained. The two phases areseparated and the organic one is distilled under reduced pressure, untila residue of approximately 50 g is obtained. This residue is dissolvedin 220 mL of ethyl acetate and the solvent distilled off under reducedpressure with an external temperature of 40° C. This operation isrepeated twice and the title compound is obtained as solid residue.

c)(S)-2-[3-(3-Fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate (IIc)

In a 2 L glass reactor 42.4 g (0.103 mol) of(S)-3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidebase are dissolved in 950 mL of ethyl acetate.

The solution is heated under stirring at 50-55° C. and kept at thistemperature for one hour. To this solution, 14.5 g (0.15 mol) ofmethanesulfonic acid are added in 20 minutes, and the temperature islowered to 20° C. in 90 minutes. After 30 minutes the solid is collectedby filtration, dried at 50° C. under reduced pressure and thencrystallized from methanol (methanol : product 1:5 by weight) to obtain25.1 g of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate, m. p. 181° C. (capillary).

¹H-NMR (DMSO-d₆) (Bruker AV300) δ (ppm, with respect to TMS): 1.44 (3H,d, J=7Hz, CH₃); 2.35 (3H, s, CH3SO₃); 3,81 (1H, q, J=7 Hz, H-2), 3.99(2H, bs, CH₂ benzylic); 4.02 (2H, AB system, CH₂N—); 5.17 (2H, s,CH₂OR); 6.98-7.63 (11H, m, aromatic H); 7.62 and 7.75 (2H, bs, NH₂amide); 9.02 (2H, broad, NH2⁺).

¹³C-NMR (DMSO-d₆) (Bruker AV300) δ (ppm): 15.9 (CH₃); 35.5 (CH₂); 39.7(CH ₃SO₃H); 48.1 (CH₂NR); 54.4(CH); 68.4(OCH₂); 112.2 (aromatic CH);112.7 (d, J_(C-F)=22Hz, aromatic CH); 113.8 (d, J_(C-F)=22Hz, aromaticCH); 114.5 (d, J_(C-F)=22 Hz, aromatic CH); 115.2 (d, J_(C-F)=22Hz,aromatic CH); 123.2 (aromatic CH); 123.8; 124.6 (aromatic CH); 128.7 and130.0 (d, J_(C-F)=6Hz, aromatic CH); 130.04 (aromatic CH); 130.3 (d,J_(C-F)=6Hz, aromatic CH); 132.6 (aromatic CH); 139.8 (d, J_(C-F)=7Hz);143.4 (d, J_(C-F)=7 Hz); 158.1, 160.5 and 163.7 (d, J_(C-F)=240, C—F);160.6 and 163.8 (d, J_(C-F)=240, C—F); 170.5 (CON). A sample (90 mg) of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamide(IIa) is isolated also by preparative HPLC from a 200 g of safinamidemethanesulfonate (Ic) prepared according to J. Med. Chem., 1998, 41,579, method A, that contains it, as methanesulfonate (IIc), in 0.12% byweight.

The separation is performed, in two stages (Stage 1 and Stage2),according to the following scheme:

Stage 1

The scope of the first stage is to isolate a crude product enriched inIIa/TFA (Trifluoroacetic acid).

Preparative HPLC conditions are reported below:

Preparative HPLC Conditions:

Instrument: Waters Delta Prep 4000 (reciprocating pump, gradientcontroller with low pressure mixer)

-   -   Radial Compression Module Prep LC Base (Waters)    -   Jasco 7125 UV-Variable detector, o.p. 0.2 mm    -   Merk D2000 printer-plotter

Column: Delta Pak C18, 15 μm, 40×100 mm (Waters)

Eluent A: 70/30, Water/Acetonitrile+0.1% TFA

Eluent B: 30/70, Water/Acetonitrile+0.1% TFA

Flow rate: 27.0 ml/min

Gradient: 40 min, isocratic 100% A, then to 100% B in 1 minute

Detection: UV 227 nm

Injection: 5 g in 50 ml of Water (by pump inlet line D)

Stage 2

This stage is needed to eliminate TFA from IIa/TFA and to further purifyIIa. IIa/TFA is chromatographed using the preparative HPLC conditionsgiven below.

The fraction 4 and 5 are combined together and evaporated at 40° C.under vacuum until complete removal of acetonitrile. The residual watersolution is kept in a refrigerator at 4° C. The insoluble is isolated byfiltration and dried under vacuum at room temperature to provide IIa (90mg; HPLC purity 100%).

Preparative HPLC Conditions:

Instrument: Waters Delta Prep 4000 (reciprocating pump, gradientcontroller with low pressure mixer)

-   -   Jasco 7125 UV-Variable detector, o.p. 0.2 mm    -   Merk D2000 printer-plotter

Column: Symmetry C18, 7 μm, 20×250 mm (Waters)

Eluent A: 70/30, Water/Acetonitrile

Eluent B: 30/70, Water/Acetonitrile

Flow rate: 15.0 ml/min

Gradient: 20 min, isocratic 100% A, then to 100% B in 10 minutes

Detection: UV 227 nm

Injection: 50 ml of impurity “IIa/TFA” solution (by pump inlet line D)

EXAMPLE 11 Preparation of(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate (IId) a) 3-(2-Fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde (Vb)

3-(2-Fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde is prepared byfollowing the same procedure of Example 10, step a) in a 1:10 scale, butusing 2-fluorobenzyl chloride instead of 3-fluorobenzyl chloride. Themolar yield is 3% with a 98.1 purity determined by GC analysis (area %,see Example 16A). The product has m.p. 71° C. (capillary).

¹H-NMR: (CDCl₃, 300 MHz, 298K) δ (ppm, with respect to TMS): 4.06 (2H,s, CH₂); 5.23 (2H, s, OCH₂); 6.95-7.40 (9H, m, aromatic H); 7.67 (1H,bd, J=0.9 Hz, aromatic H ortho to C═O and CH₂); 7.76 (1H, dd, J₁=2.1 Hz,J₂=8.3 Hz, aromatic H ortho to C═O and aromatic CH); 9.84 (1 H, s, CHO).

¹³C-NMR: (CDCl₃, 75.4 MHz, 298K) δ (ppm): 29.2 (CH₂); 64.1 (OCH₂); 111.4(aromatic CH); 115.4 (d, J_(C-F)=22.0 Hz, aromatic CH), 115.5 (d,J_(C-F)=21.1 Hz, aromatic CH); 123.3 (d, J_(C-F)=14.2 Hz, quaternaryaromatic C); 124.1 (d, J_(C-F)=2.6 Hz, aromatic CH); 124.5 (d,J_(C-F)=3.2 Hz, aromatic CH); 126.6 (d, J_(C-F)=15.5 Hz, quaternaryaromatic C); 128.2 (d, J_(C-F)=8.1 Hz, aromatic CH); 129.6 (d,J_(C-F)=6.2 Hz, aromatic CH); 129.6 (quaternary aromatic C); 130.0(quaternary aromatic C); 130.2 (d, J_(C-F)=8.3 Hz, aromatic CH); 131.1(aromatic CH); 131.3 (d, J_(C-F)=4.1 Hz, aromatic CH); 131.8 (aromaticCH); 160.5 (d, J_(C-F)=246.8 Hz, quaternary aromatic C); 161.2 (d,J_(C-F)=245.1 Hz, quaternary aromatic C); 161.3 (quaternary aromatic C);191.1 (CHO).

b)(S)-2-[3-(2-Fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamide(IIb)

(S)-2-[3-(2 -Fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]-propanamide is prepared by following thesame procedure of Example 10, step b) by using3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde instead of3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde. The yield is 83%;m.p. 161° C. (capillary).

¹H--NMR: (CDCl₃, 300 MHz, 298K) δ (ppm, with respect to TMS): 1.32 (3H,d, J=6.7 Hz, CH₃); 1.97 (1H, bs, NH); 3.22 (1H, q, J=6.7 Hz, CH—CO);3.67 (2H, ABq, J=12.8 Hz, diastereotopic H of NCH₂); 4.03 (2H, s, CH₂);5.12 (2H, s, OCH₂); 5.98 (1H, bs, NH₂); 6.89 (1H, d, J_(orto)=8.3 Hz,aromatic H ortho to CH₂NH and aromatic CH); 6.95-7.40 (10H, m, aromaticH).

¹³C-NMR: (CDCl₃, 75.4 MHz, 298K) δ (ppm): 19.6 (CH₃); 29.2 (CH₂); 52.0(NHCH₂); 57.7 (H₂NCOCH); 63.8 (OCH₂); 111.7 (aromatic CH); 115.2 (d,J_(C-F)=21.9 Hz, aromatic CH), 115.3 (d, J_(C-F)=21.3 Hz, aromatic CH);124.0 (d, J_(C-F)=3.5 Hz, aromatic CH); 124.3 (d, J_(C-F)=2.9 Hz,aromatic CH); 124.3 (d, J_(C-F)=14.4 Hz, quaternary aromatic C); 127.5(aromatic CH); 127.6 (d, J_(C-F)=15.0 Hz, quaternary aromatic C); 127.8(d, J_(C-F)=7.5 Hz, aromatic CH); 128.8 (quaternary aromatic C);129.0-130.0 (m, 2 aromatic CH); 130.5 (aromatic CH); 131.3 (d,J_(C-F)=4.6 Hz, aromatic CH); 131.8 (quaternary aromatic C); 155.6(quaternary aromatic C); 160.4 (d, J_(C-F)=245.8 Hz, quaternary aromaticC); 161.2 (d, J_(C-F)=244.6 Hz, quaternary aromatic C); 178.2 (CONH₂).

c)(S)-2-[3-(2-Fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate (IId)

(S)-2-[3-(2-Fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate is prepared by following the same procedure of Example10, step c) but using(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideas the starting material. The yield is 89%; m.p. 190° C. (capillary).

¹H-NMR: (DMSO-d₆, 300 MHz, 298K) δ (ppm, with respect to TMS): 1.42 (3H,d, J=6.8 Hz, CH ₃CH); 2.33 (3H, s, CH₃SO₃); 3.50-4.20 (5H, m, CH—CO,CH₂, diastereotopic H of _(NCH) ₂,); 5.19 (2H, s, OCH₂); 6.95-8.00 (11H,m, aromatic H); 9.02 (2H, bs, NH₂+).

¹³C-NMR: (DMSO-d₆, 75.4 MHz, 298K) δ (ppm): 16.5 (CH3); 28.8 (CH₂); 48.6(NHCH₂); 54.9 (H₂NCOCH); 64.3 (OCH₂); 112.8 (aromatic CH); 115.0-117.0(2 aromatic CH); 124.2 (d, J_(C-F)=14.4 Hz, quaternary aromatic C);124.4 (quaternary aromatic C); 124.8 (aromatic CH); 125.0 (aromatic CH);127.3 (d, J_(C-F)=16.1 Hz, quaternary aromatic C); 128.6 (quaternaryaromatic C); 128.8 (aromatic CH); 129.0-133.0 (m, 5 aromatic CH); 156.9(quaternary aromatic C); 160.8 (d, J_(C-F)=245.2 Hz, quaternary aromaticC); 160.9 (d, J_(C-F)=243.5 Hz, quaternary aromatic C); 171.1 (CONH₂).

EXAMPLE 12 Preparation of(S)-2-[4-3-fluorobenzyloxy)benzylamino]propanamide (safinamide)methanesulfonate (Ic) from 4-(3-fluorobenzyloxy)benzaldehyde (IVa)contaminated by 1% by weight of impurity3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde (Va)

To 4-(3-fluorobenzyloxy)benzaldehyde (10 g; GC purity 98.8, area %), 1%of 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde is added and themixture is converted into(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide safinamide base byfollowing the same procedure of Example 6. The yield is 84% with acontent of impurity (IIa) of 0.84% (see Example 17B) by weight. The freebase (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (Ia) isconverted into the corresponding methanesulphonate by following the sameprocedure of Example 7 to provide the methanesulphonate (Ic) in 98%yield with a content of impurity(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate (IIc) of 0.62% by weight determined by HPLC (seeExample 17B).

EXAMPLE 13

Crystallization of safinamide methanesulfonate (Ic) Contaminated byImpurity (IIc)

The safinamide methanesulfonate contaminated by(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate (IIc) of 0.62% by weight determined by HPLC (seeExample 17B) obtained according to Example 12, is crystallized by usingfive different(s) solvent systems by dissolving at reflux temperatureand cooling at room temperature.

The result are reported in the following Table 5

TABLE 5 % w/w of IIc in Ic after % TEST SOLVENT SYSTEM ANDcrystallization Molar No. AMOUNT (mL/g) (*) Yield 13a 2-PrOH/MeOH 2:1,45 0.28 44.9 13b EtOAc/MeOH 4:1, 50 0.15 29.6 13c EtOH, 10 0.30 73.2 13dAcetone/H₂O ~27:1, 40.5 0.08 20.6 13e Acetonitrile/H₂O 60:1, 30.5 0.0969.3 (*) the % (w/w) is evaluated according to Example 17B.

EXAMPLE 14 Preparation of(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (safinamide, Ia)methanesulfonate (Ic) According to Known Methods 14.1 Preparation of4-(3-fluorobenzyloxy)benzaldehyde (IVa) 14.1.a) Procedure of Example 1aof U.S. Pat. No. 6,335,354 B2

4-(3-Fluorobenzyloxy)benzaldehyde (IVa) is prepared by the proceduredescribed in Example 1a of U.S. Pat. No. 6,335,354 B2.

Accordingly, a mixture of 3-fluorobenzyl chloride (2.86 g, 19.80 mmol)4-hydroxybenzaldehyde (3.03 g, 24.80 mmol), K₂CO₃ (10.30 g, 74.50 mmol),NaI (137.1 mg, 0.91 mmol), and ethanol, (40 mL) is heated to reflux in70 minutes and kept at reflux temperature for 4 hours and 15 minutes.

After working up the reaction mixture,4-(3-fluorobenzyloxy)benzaldehyde, is isolated as a yellow oil in 95%yield.

The product has GC purity of 97.6 (area %, see Example 16A) and acontent of 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde (Va) of0.14% by weight determined by GC (see Example 16B)

14.1.b) Procedure of J. Agric. Food Chem, 27, 4, 1979

4-(3-Fluorobenzyloxy)benzaldehyde (IVa) is prepared by the procedurereported in J. Agric. Food Chem, 27, 4, 1979.

Accordingly, 3-fluorobenzyl chloride (14.5 g, 100 mmol) is added understirring and under nitrogen atmosphere to a solution of4-hydroxybenzaldehyde (12.2 g, 100 mmol) and of NaOH (4.0 g, 100 mmol)in ethanol (100 mL).

The mixture is gradually heated in 25 minutes to reflux and stirred atreflux temperature for 6 hours and 20 minutes. The reaction mixture isfiltrated and then concentrated at reduced pressure to obtain4-(3-fluoro-benzyloxy)benzaldehyde (23.43 g) as a yellow solid residue.Dichloromethane (250 mL) is added to the residue, the insoluble isfiltered and the resulting solution is concentrated under reducedpressure to provide 4-(3-fluorobenzyloxy)benzaldehyde as a yellow solid,in 80.4% yield.

The product has GC purity of 91.6 (area %, see Example 16A) and acontent of 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde (Va) of0.13% by weight determined by GC(see Example 16B)

14.2 Preparation of (S)-2-[4-(3-fluorobenzyloxy)benzylamino] propanamide(Ia) and its methanesulfonate salt (Ic) 14.2.a) Procedure of J. Med.Chem., 1998, 41, 579, Method A

(S)-2-[4-(3-Fluorobenzyloxy)benzylamino]propanamide (Ia) is prepared byreacting 4-(3-fluorobenzyloxy)benzaldehyde (10 mmol), prepared asdescribed in Example 14.1.a., and L-alaninamide hydrochloride (1.37 g,11 mmol) followed by reduction with NaBH₃CN(0.50 g, 8 mmol). Afterworking up the reaction mixture and purification byflash-chromatography, (S)-2[4-(3-fluorobenzyloxy)benzylamino]propanamideis isolated as white solid in 68.7% yield. The product has HPLC purityof 96.2 (area %, see Example 17A) and a content of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamide(IIa) of 0.15% by weight (see Example 17B).

A mixture of (S)-2[4-(3-fluorobenzyloxy)benzylamino] propanamide (1.50g, 4.96 mmol) and ethyl acetate (40.2 mL) is heated to 50° C. until aclear solution is obtained. Methanesulfonic acid (0.53 g, 5.51 mmol) isadded under stirring in 15 minutes to the solution and the resultingheterogeneous mixture is cooled under stirring to 20° C. in 90 minutes.After 30 minutes at 20° C. the solid is collected by filtration, washedwith ethyl acetate (6 mL) and dried at 50° C. at reduced pressure for 15hrs to provide (S)-2[4-(3-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate (Ic) as a white solid in a 96.1% yield. The product hasHPLC purity 98.6 (area %, see Example 17A) and a content of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate (IIc) of 0.10% by weight determined by HPLC (seeExample 17B).

14.2.b) Procedure of J. Med. Chem., 1998, 41, 579, Method A

(S)-2-[4-(3-Fluorobenzyloxy)benzylamino]propanamide (Ia) is preparedaccording to example 14.2.a from 4-(3-fluorobenzyloxy)benzaldehyde (10mmol), prepared as described in example 14.1.b., and L-alaninamidehydrochloride (1.37 g, 11 mmol) followed by reduction with NaBH₃CN(0.50g, 8 mmol).

(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (Ia), is obtained aswhite solid in 66.5% yield. The product has HPLC purity of 88.5 (area %,see Example 17A) and a content of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamide(IIa) of 0.064% by weight determined by HPLC (see Example 17B).(S)-2-[4-(3-Fluorobenzyloxy)benzylamino]propanamide (Ia) is convertedinto the corresponding methanesulfonate (Ic) in a 88.9% yield bytreatment with methanesulfonic acid according to Example 14.2.a. Theproduct has a HPLC purity of 97.7 (area %, see Example 17A) and acontent of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamidemethanesulfonate (IIc) of 0.05% by weight determined by HPLC (seeExample 17B).

EXAMPLE 15 Preparation of(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (ralfinamide, Ib)methanesulfonate (Id) According to Known Methods 15.1 Preparation of4-(2-fluorobenzyloxy)benzaldehyde (IVb) 15.1.a) Procedure of Example 1aof U.S. Pat. No. 6,335,354 B2

4-(2-Fluorobenzyloxy)benzaldehyde (IVb) is prepared according to theExample 14.1.a) from 2-fluorobenzyl chloride (14.3 g, 98 mmol),4-hydroxybenzaldehyde (15.1 g, 123 mmol), K₂CO₃ (51 g, 369 mmol), NaI(500 mg, 3.3 mmol.) ethanol, 75 mL.

The mixture is kept at reflux for 12 hrs. After working up the reactionmixture, 4-(2-fluorobenzyloxy)benzaldehyde is obtained in 75% yield as ayellow oil. The product has GC purity of 92.1 (area %, see Example 16A)and a content of 3-(2-fluorobenzyl)4-(2-fluorobenzyloxy)benzaldehyde of0.25% by weight determined by G.C. (see Example 16B).

15.1.b) Procedure of J. Agric. Food Chem, 27, 4, 1979

4-(2-Fluorobenzyloxy)benzaldehyde (IVb) is prepared according to Example14.1.b from 2-fluorobenzyl chloride (18.0 g, 123 mmol),4-hydroxy-benzaldehyde (15.3 g, 125 mmol), NaOH (5.0 g, 12 mmol) andethanol (125 mL).

The mixture is heated in 25 minutes to reflux and kept at refluxtemperature under stirring for 12 hours.

After working up the reaction mixture according to Example 14.1.b4-(2-fluorobenzyloxy)benzaldehyde is obtained as a yellow solid, in90.0% yield. The product has GC purity of 90.4 (area %, see Example 16A)and a content of 3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde(Vb) of 0.14% by weight determined by G.C. (see Example 16B).

15.2 Preparation of (S)-2-[4-(2-fluorobenzyloxy)benzylamino] propanamide(Ib) and its methanesulfonate salt (Id) 15.2.a) Procedure of J. Med.Chem, 1998, 41, 579, Method A

(S)-2-[4-(2 -Fluorobenzyloxy)benzylamino]propanamide (Ib) is preparedfollowing the procedure of Example 14.2.a by using4-(2-fluorobenzyloxy)benzaldehyde (10 mmol, prepared as in Example 15.1a) instead of 4-(3-fluorobenzyloxy)benzaldehyde.

(S)-2[4-(2-Fluorobenzyloxy)benzalamino]propanamide is obtained in 67.3%yield as a white solid. The product has a HPLC purity of 86.7 (area %,see Example 17A) and a content of(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamide(IIb) of 0.22% by weight determined by HPLC (see Example 17B).

A mixture of (S)-2[4-(2-fluorobenzyloxy)benzylamino]propanamide (1.50 g,4.96 mmol) and propan-2-ol (10.5 mL) is heated to 50° C. and kept atthis temperature until a clear solution is obtained. Methanesulfonicacid, (0.48 g, 5.01 mmol) is added under stirring in 15 minutes.

The heterogeneous mixture is then cooled under stirring to 20° C. in 2hours. After 1 hour at 20° C. the solid is collected by filtration,dried at reduced pressure to provide(S)-2[4-(2-fluorobenzyloxy)benzylamino] propanamide methanesulfonate aswhite solid in 89.1% yield. The product has a HPLC purity of 96.9 (area%, see Example 17A) and a content of(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate (IId) of 0.14% by weight determined by HPLC (seeExample 17B).

15.2.b) Procedure of J. Med. Chem. 1998, 41, 579, Method A

(S)-2- [4- (2 -Fluorobenzyloxy)benzylamino]propanamide (Ib) is preparedaccording to Example 14.2.b by using 4-(2-fluorobenzyloxy)benzaldehyde(10 mmol, prepared according to Example 15.1.b) instead of4-(3-fluorobenzyloxy)benzaldehyde.

(S)-2-[4-(2-Fluorobenzyloxy)benzylamino]propanamide is obtained as awhite solid in 58.8% yield. The product has a HPLC purity 83.8 (area %,see Example 17A) and a content of(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamide(IIb) of 0.15% by weight determined by HPLC (see Example 17B).

(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (Ib) is convertedinto the corresponding methanesulfonate (Id) in a 89.4% yield as a whitesolid. The product has a HPLC purity of 95.2 (area %, see Example 17A)and a content of

(S) -2-[3-(2 -fluorobenzyl) -4-(2-fluorobenzyloxy)-benzylamino]propanamide methanesulfonate of 0.11% byweight determined by HPLC (see Example 17B).

EXAMPLE 16A GC determination of 4-(3-fluorobenzyloxy)benzaldehyde and4-(2-fluorobenzyloxy)benzaldehyde Purity

Test Preparation

Dissolve about 100 mg of the sample in 10 ml of methylene chloride.Chromatographic conditions

The chromatographic procedure is carried out by using:

-   -   a fused silica capillary column 60 m long and 0.32 mm internal        diameter. RTX 35 (35% Diphenyl- 65% Dimethyl polysiloxane) Film        thickness=0.25 μm;    -   helium as carrier gas at a pressure of 150 kPa;    -   a split flow of 25 ml/min;    -   injector temp. 290° C.;    -   detector (FID) temp. 290° C.;

with the following temperature program:

Time Temperature Rate (min) (° C.) (° C./min) Comment 0-5 150 —isothermal  5-11 150→240 15 linear gradient 11-19 240 — isothermal  19-20.7 240→290 30 linear gradient 20.7-40   290 — isothermal

Procedure

Inject 1 μl of the Test Preparation. Record the chromatogram andcalculate the product purity by area percent calculation.

Impurities Identification

4-(3-Fluorobenzyloxy)benzaldehyde (IVa):

Retention Times:

4-(3-Fluorobenzyloxy)benzaldehyde retention time is about 17.

4-Hydroxybenzaldehyde relative retention time is about 0.52.

4-(2-Fluorobenzyloxy)benzaldehyde relative retention time is about 0.98.

4-(4-Fluorobenzyloxy)benzaldehyde relative retention time is about 1.01.

4-Benzyloxybenzaldehyde relative retention time is about 1.02.

3-(3-Fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde relative retentiontime is about 1.78.

4-(2-Fluorobenzyloxy)benzaldehyde (IVb):

Retention Times:

4-(2-Fluorobenzyloxy)benzaldehyde retention time is about 17.

4-Hydroxybenzaldehyde relative retention time is about 0.53.

4-(3-Fluorobenzyloxy)benzaldehyde relative retention time is about 1.02.

4-(4-Fluorobenzyloxy)benzaldehyde relative retention time is about 1.03.

4-Benzyloxybenzaldehyde relative retention time is about 1.04.

3-(2-Fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde relative retentiontime is about 1.81.

EXAMPLE 16B GC determination of the Content of3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde (Vb) in4-(2-fluorobenzyloxy) benzaldehyde (IVb) and of3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy) benzaldehyde (Va) in4-(3-fluorobenzyloxy)benzaldehyde (IVa)

The known related substance taken into consideration for4-(2-fluorobenzyloxy)benzaldehyde is the3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde and for4-(3-fluorobenzyloxy)benzaldehyde is the3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde. The determinationis carried out according to the following conditions:

Internal Standard Solution

Prepare a 3,4,5-trimethoxybenzaldehyde solution with concentration 1.5mg/ml in methylene chloride (IS).

Reference Solution for the 3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde Determination in the 4-(2-fluorobenzyloxy)benzaldehyde:

Accurately weigh about 20 mg of3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde reference standardand 20 mg of 4-(2-fluorobenzyloxy)benzaldehyde reference standard in a20 mL volumetric flask, dissolve and dilute to volume with diluent;transfer 500 μL of this solution in a 5 mL volumetric flask, add 500 μLof IS solution and dilute to volume with diluent to obtain a solutioncontaining 3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde and4-(2-fluorobenzyloxy)benzaldehyde at about 100 μg/mL (corresponding toabout 0.10%).

Reference Solution for the 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde Determination in the 4-(3-fluorobenzyloxy)benzaldehyde:

Accurately weigh about 20 mg of3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde reference standardand 20 mg of 4-(3-fluorobenzyloxy)benzaldehyde reference standard in a20 mL volumetric flask, dissolve and dilute to volume with diluent;transfer 500 μL of this solution in a 5 mL volumetric flask , add 500 μLof IS solution and dilute to volume with diluent to obtain a solutioncontaining 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde and4-(3-fluorobenzyloxy)benzaldehyde at about 100 μg/mL (corresponding toabout 0.10%).

Test Solution:

Accurately weigh about 500 mg of test product in a 5 mL volumetricflask, add 500 μL of IS solution, dissolve and dilute to volume withdiluent to obtain a solution having known concentration of about 100mg/mL.

Chromatographic Conditions:

The chromatographic procedure is carried out by using:

-   -   Column: a fused silica capillary column RTX 35 (35% Diphenyl-65%        Dimethyl polysiloxane) 60 m long, 0.32 mm I.D. , film thickness        0.25 μm;    -   Carrier (helium) at pressure of 150 kPa;    -   Split flow 25 mL/min;    -   Injector temp. 290° C.;    -   Detector (FID) temp. 290° C.;    -   Temperature program: 0-5 min isothermal at 150° C. , 5-11 min        linear from 150° C. to 240° C. at a rate of 15° C./ min , 11-19        min isothermal at 240° C. , 19-21 min linear from 240° C. to        290° C. at a rate of 30° C/min , 21-40 min isothermal at 290°        C.;    -   diluent: methylene chloride    -   injection volume 1 μL.

Procedure:

Inject blank (diluent), reference solution, test solution and record thechromatograms.

In the reference chromatogram verify that:

4-(2-Fluorobenzyloxy)benzaldehyde retention time is about 18 min;

3-(2-Fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde relative retentiontime is about 1.7

or

4-(3-Fluorobenzyloxy)benzaldehyde retention time is about 18 min;

3-(3-Fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde relative retentiontime is about 1.7

3,4,5-Trimethoxybenzaldehyde (IS) relative retention time is about 0.7

Calculate the percent content of3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde in the4-(2-fluorobenzyloxy)benzaldehyde examined or of the3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde in the4-(3-fluorobenzyloxy)benzaldehyde examined by internal standardcalculation. The value of the limit of quantitation (LOQ) for(3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde and of3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde is 0.005% byweight. The value of the limit of detection (LOD) for both consideredimpurities is 0.0025% by weight.

EXAMPLE 17A HPLC Determination of Purity of (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (safinamide (Ia)), its methanesulfonate (Ic),(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (ralfinamide (Ib))and its methanesulfonate (Id).

The following chromatographic procedure is suitable for both the freebase form (Ia, Ib) and the methanesulfonate salt (Ic, Id) of theproducts.

Diluent

Mobile phase.

Test solution

Accurately weigh about 25 mg of product in a 25 ml volumetric flask,dissolve in and dilute to volume with diluent to obtain a solutionhaving known concentration of about 1.0 mg/ml.

Chromatographic Condition

The chromatographic procedure is carried out by using:

-   -   Column: Waters Symmetry C8, 150×4.6 mm, 5μ;    -   detection: UV 220 nm;    -   column temperature: 30° C.    -   mobile phase: 40% solvent A+10% solvent B+50% solvent C,        containing 1.0 g/l sodium octansulphonate;        -   solvent A: Buffer solution =KH₂PO₄ 0.05M;        -   solvent B: Acetonitrile;        -   sovent C: Methanol;    -   isocratic elution, run time: 60 minutes;    -   flow rate: 1.0 ml/min;    -   injection volume: 10 μl.

Procedure

Inject the test solution, record the chromatogram and calculate theproduct purity by area percent calculation.

(S)-2-[4-(3-Fluorobenzyloxy)benzylamino]propanamide (safinamide) andrelated impurities identification

Retention Time:

(S)-2-[4-(3-Fluorobenzyloxy)benzylamino]propanamide retention time isabout 5.5 min.

(S)-2-[4-(3-Fluorobenzyloxy)benzylamino]propionic acid relativeretention time is about 0.73.

(S)-2-[3-(3-Fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamiderelative retention time is about 4.08.

(S)-2-[4-(2-Fluorobenzyloxy)benzylamino]propanamide (ralfinamide) andrelated impurity identification

Retention Time:

(S)-2-[4-(2-Fluorobenzyloxy)benzylamino]propanamide retention time isabout 5.5 min.

(S)-2-[4-(2-Fluorobenzyloxy)benzylamino]propionic acid relativeretention time is about 0.73.

(S)-2-[3-(2 -Fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamide relative retention time isabout 4.08.

EXAMPLE 17B HPLC Determination of(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy) benzylamino]propanamide(free base, IIb and methanesulfonate, IId) in(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (free base, Ib andmethanesulfonate, Id) and of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamide(free base, IIa and methanesulfonate, IIc) in(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (free base, Ia andmethanesulfonate, Ic)

The determination of the (S)-2-[3-(2 -fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamide (free base andmethanesulfonate) in (S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide(free base and methanesulfonate) samples and of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamide(free base and methanesulfonate) in(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (free base andmethanesulfonate) samples is carried out according to the followingconditions:

Reference Solution for the(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamideDetermination in the(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide:

Accurately weigh about 30 mg of(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate reference standard and 20 mg of(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide reference standardin a 50 mL volumetric flask , dissolve and dilute to volume withdiluent; dilute 1.0 mL of this solution to 20 mL with diluent (1^(st)dilution); dilute 1.0 mL of the last solution to 20 mL with diluent(2^(nd) dilution) to obtain a solution containing 2-[3-(2-fluorobenzyl)-4-(2 -fluorobenzyloxy)benzylamino]propanamide (about0.12%) at about 1.20 μg/ mL and (S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide methanesulfonate at about 1.00μg/mL (about 0.10%).

Reference Solution for the(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate Determination in the(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide methanesulfonate:

Accurately weigh about 30 mg of(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate reference standard and 20 mg of(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide methanesulfonatereference standard in a 50 mL volumetric flask , dissolve and dilute tovolume with diluent ; dilute 1.0 mL of this solution to 20 mL withdiluent (1st dilution); dilute 1.0 mL of the last solution to 20 mL withdiluent (2^(nd) dilution) to obtain a solution containing2[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamide (about0.15% as methanesulfonic salt) at about 1.20 μg/ mL and(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide methanesulfonate atabout 1.00 μg/mL (about 0.10%).

Reference Solution for the(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamidein the (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide:

Accurately weigh about 24 mg of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamidereference standard and 20 mg of(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide reference standardin a 50 mL volumetric flask , dissolve and dilute to volume with diluent; dilute 1.0 mL of this solution to 20 mL with diluent (1st dilution);dilute 1.0 mL of the last solution to 20 mL with diluent (2nd dilution)to obtain a solution containing2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamide(about 0.12%) at about 1.20 μg/mL and(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide methanesulfonate atabout 1.00 μg/mL (about 0.10%).

Reference Solution for the(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate in the(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide methanesulfonate:

Accurately weigh about 24 mg of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamidereference standard and 20 mg of (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide methanesulfonate referencestandard in a 50 mL volumetric flask , dissolve and dilute to volumewith diluent; dilute 1.0 mL of this solution to 20 mL with diluent (1stdilution); dilute 1.0 mL of the last solution to 20 mL with diluent(2^(nd) dilution) to obtain a solution containing2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamide(about 0.15% as methanesulfonic salt) at about 1.20 μg/mL and(S)-2-[4-(3 -fluorobenzyloxy)benzylamino]propanamide methanesulfonate atabout 1.00 μg/mL (about 0.10%).

Test Solution:

Accurately weigh about 25 mg of test product in a 25 mL volumetricflask, dissolve and dilute to volume with diluent to obtain a solutionhaving known concentration of about 1.0 mg/mL.

Chromatographic Conditions:

The chromatographic procedure is carried out by using:

-   -   Column: Waters Simmetry C8 150×4.6 mm, 5μ, or equivalent    -   column temperature: 30° C.    -   mobile phase: mixture of 40% solvent A: 10% solvent B: 50%        solvent C, containing 1 g/lt of sodium octanesulfonate    -   solvent A: buffer solution 0.05M KH₂PO₄;    -   solvent B: acetonitrile;    -   solvent C: methanol;    -   isocratic elution;    -   run time: 60 min;    -   flow rate: 1.0 mL/min;    -   detection: UV 220 nm;    -   injection volume: 100 μl;    -   diluent: mobile phase

Procedure:

Inject blank (diluent), reference solution, test solution and record thechromatograms.

In the reference chromatogram verify the following system suitabilityparameters:

(S)-2-[4-(2-Fluorobenzyloxy)benzylamino]propanamide retention time isabout 5.2 minutes;

The USP tailing for (S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamidepeak is in the range between 0.8 and 1.5;

(S)-2-[3-(2-Fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamiderelative retention time is about 5.1.

or

(S)-2-[4-(3-Fluorobenzyloxy)benzylamino]propanamide retention time isabout 5.5 minutes;

The USP tailing for (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamidepeak is in the range between 0.8 and 1.5;

(S)-2-[3-(3-Fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamiderelative retention time is about 4.1.

Adjust the mobile phase in order to obtain the system suitability.

Calculate the percent content(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamide(free base and methanesulfonate) in the examined(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (free base andmethanesulfonate) samples and of(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamide(free base and methanesulfonate) in the examined(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (free base andmethanesulfonate) samples by external standard calculation.

The value of the limit of quantitation (LOQ) for(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamideandfor(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamidein the corresponding (S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamideand (S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide is 0.004% byweight.

The value of the limit of quantitation (LOQ) for(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate andfor(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate in the corresponding(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide methanesulfonate and(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide methanesulfonate is0.005% by weight. The value of the limit of detection for all theconsidered impurities is 0.001% by weight.

EXAMPLE 18 HPLC Determination of(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (ralfinamide)methanesulfonate (Id) enantiomeric Purity

The enantiomeric purity of the sample is evaluated by HPLC. Thedetermination is carried out according to the following:

Standard Solution 1:

Dissolve about 5.3 mg of(R)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide methanesulfonatereference standard in 25 mL of mobile phase.

Standard Solution 2:

Dissolve about 8.0 mg of(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide methanesulfonatereference standard and 0.2 mL of standard solution 1 in 50 mL of mobilephase.

The concentration of (R)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate is about 0.5% calculated with respect to theconcentration of (S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate.

Test Solutions 1 and 2:

In duplicate, dissolve about 8.0 mg of the test product in 50 mL ofmobile phase.

Chromatographic Conditions:

-   -   Column: Chiralpak WH 250 mm×4.6 mm, I.D. 5 μm;    -   column temperature: 45° C.;    -   mobile phase: 0.25 mM CuSO₄ (accurately weigh about 40 mg of        CuSO₄ in 1000 mL of water)/MeOH 60/40;    -   isocratic elution;    -   flow rate: 1.0 mL/min;    -   detection: UV 230 nm;    -   injection volume: 10 μl;    -   run time: 15 minutes.

Procedure:

Analyse blank (mobile phase) once, standard solution 2 twice, testsolutions 1 and 2 once and verify that:

-   -   for the standard injections, the RSD % for        (R)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide        methanesulfonate percent area is less than 2.0%;    -   both for standard and sample solutions, for each injection the        main peak percent area is included between the average value        ±0.1%.

Calculate the (R)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamidemethanesulfonate content (percent area) as mean of the twodetermination.

Retention Times:

(S)-2-[4-(2-Fluorobenzyloxy)benzylamino]propanamide retention time isabout 5.7 min.

(R)-2-[4-(2-Fluorobenzyloxy)benzylamino]propanamide relative retentiontime is about 1.7.

EXAMPLE 19 HPLC Determination of(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (safinamide)methanesulfonate (Ic) enantiomeric Purity

The enantiomeric purity of the sample is evaluated by HPLC. Thedetermination takes place according to the following conditions:

Test Solution:

Dissolve about 10 mg of test sample in 10 mL of mobile phase.

Chromatographic Conditions:

-   -   Column: Chiralpak WH 250mm×4.6 mm, I.D. 10 μm;    -   column temperature: 50° C.;    -   mobile phase: 0.25 mM CuSO₄    -   isocratic elution;    -   flow rate: 1.0 mL/min;    -   detection: UV 200 nm;    -   injection volume: 10 μl;    -   run time: 30 minutes.

Procedure:

Inject the test solution and calculate the enantiomers peak response asarea percent.

(S)-2-[4-(3-Fluorobenzyloxy)benzylamino]propanamide retention time isabout 9.2 min.

(R)-2-[4-(3-Fluorobenzyloxy)benzylamino]propanamide relative retentiontime is about 1.9.

EXAMPLE 20 Cytochrome P450 Assay

Inhibition of the five most important Cytochrome P450 isoforms (CYP1A2,CYP2C9, CYP2C19, CYP2D6 and CYP3A4), involved in drug metabolism, wasmeasured using specific substrates that become fluorescent upon CYPmetabolism (Gentest Kit assay).

Compounds were tested in a 96-well plate containing incubation/NADPHregenerating buffer. Specific human recombinant isoenzymes andsubstrates were added and incubated at 37° C. for 15 minutes forCYP1A2/CEC, 40 minutes for CYP2E1 /MFC, 45 minutes for CYP2C9/MFC and 30minutes for the others CYP450.

The specific substrates were the following: 3-cyano-7-ethoxycoumarin(CYP2C19 and CYP1A2),

7-methoxy-4-trifluoromethylcoumarin (CYP2C9),

3[2(N,N-diethyl-N-mwthylamino) ethyl]-7-methoxy-4-methylcoumarin(CYP2D6) benzylphenylcoumarin (CYP3A4)

The plates were read on a Victor plate reader (Perkin Elmer) at theappropriate emission/excitation wavelengths, and the IC₅₀ (concentrationinhibiting by 50% the enzyme activity) determined. The results arereported in Tables 1 and 2.

EXAMPLE 21 Cytotoxicity Assay in Human Neuroblastoma Cell Line SH-SY-5Y

At time zero, the cells were seeded at 1.10⁴/cm² in 96 well plates inDMEM growth medium+10% heat inactivated FBS+2 mM 1-Glutamine+100 U/mL-100 μg/mL Penicillin/ Streptomycin.

After 72 hours at subconfluent phase of growth, the medium was removedand cells were incubated for 24 hours at 37° C. in 180 μl of neurobasalmedium+2 mM 1-Glutamine (Life Technologies) with or without testcompounds (20 μl, at least 5 concentrations in triplicate).

At the end of incubation, 20 μl of Alamar Blue dye (AlamarBlue™ AssayKit, Promega) were directly added to the cell medium.

Four hours after, the cytotoxicity was assessed by measuring thefluorescence at 530 nm excitation and 595 nm emission using TecanSpectrafluor plate reader.

Before and at the end of the treatment, the cultures were monitoredmicroscopically by an Olympus IX70 inverted light microscope matched toan Image Analyzer (Image Pro Plus, 5.1) to evaluate the cellularmorphology. Results are expressed in Table 1 as concentration inducing50% of mortality.

EXAMPLE 22 HERG Current in Transfected CHO Cell Lines

The inhibition of HERG current was tested in CHO cells stably expressingrecombinant HERG channel.

To evaluate the effect of the test compounds on HERG currents, cellswere clamped at −80 Mv, depolarised to 0 mV for 5 seconds allowingactivation of HERG current and repolarised to −50 mV during 5 secondsallowing HERG tail current to deactivate. This procedure was repeated ata frequency of 0.06 Hz. The current amplitude upon repolarisation (HERGtail current) was measured before and after exposure to the testcompound.

Inhibition of current was calculated as the difference between theamplitude of HERG tail current amplitude measured at the end of externalbath perfusion period and HERG tail current measured at the end of testcompound perfusion period (when steady-state effect is reached) dividedby control HERG tail current.

Drug concentration-inhibition curves were obtained by plotting tonicblocks versus drug concentrations. Dose-response curves were fitted tothe tonic block data, according to the logistic equation:y=A2+(A1−A2)/[1+(x/IC₅₀)^(P)]. A1 and A2 are fixed values of 0 and 1corresponding to 0 and 100% current inhibition, x is the drugconcentration, IC₅₀ is the drug concentration resulting in 50% currentinhibition and p is the corresponding slope factor. The results arereported in Table 1.

EXAMPLE 23 Maximal Electroshock Test (MES) in Mice

The maximal electroshock test (MES) is used commonly in the screening ofanti-epileptic drugs in rodent models.

Animals and Apparatus: Male CD1 mice weighing 25 g were used. Theprocedure described by White et al. (White H. S., Woodhead J. H.,Franklin M. R., Swinyard E. A., and Wolf H. H. Antiepileptic Drugs(1995) 4^(th) ed.: 99-110, Raven Press, Ltd., New York) was followed. AnUgo Basile electroconvulsive generator (Model ECT UNIT 7801) was used todeliver an electrical stimulus sufficient to produce a hindlimb tonicextensor response in at least 97% of control animals. The stimulus wasdelivered intra-aurally through clip electrodes in mice (0.7 seconds ofa 40 mA shock, with a pulse train of 80 Hz having a pulse duration of0.4 ms). The acute effect of compounds administered intraperitoneally ororally 15-60 minutes before MES induction were examined and comparedwith a vehicle control group. Ten mice were studied per group. Completesuppression of the hindlimb tonic extensor component of seizures wastaken as evidence of anticonvulsant activity.

The compounds of the invention were administered orally orintraperitoneally at the doses of 3-30 mg/kg.

The results are expressed in Tables 3 and 4 as % of protection.

EXAMPLE 24 Amphetamine Plus Chlordiazepoxide-induced Hyperlocomotion inMice

The anti mania activity, predictive of efficacy in bipolar disorders,was measured using the “Amphetamine plus chlordiazepoxide-inducedhyperlocomotion in mice” model according to the following procedure.

In this model, mice are treated with a mixture of d-amphetamine plus ananxiolytic dose of the benzodiazepine, chlordiazepoxide (Rushton R andSteinberg H. Nature 1966; 211: 1312-3; R. Arban, et al,. BehaviouralBrain Research, 158: 123-132). The model has been claimed to mimic someaspects of mania in bipolar disorder. Importantly, the hyperactivityinduced by the mixture of d-amphetamine and chlordiazepoxide could beprevented by prior administration of the established mood stabilizer,lithium, as well as other mood stabilizers drugs (e.g. magnesiumvalproate and carbamazepine). Therefore, this model has face andpredictive validity as a model of bipolar disorders and represents avaluable tool to determine, if a test compound could be a potential moodstabilizer drug candidate.

D-amphetamine (Amph) (2.5 mg/kg ip) plus chlordiazepoxide hydrochloride(CDP) (3 mg/kg ip) were administered to male Albino Swiss mice (25-32 g)in a volume of 10 ml/kg. The locomotor activity was recorded using anautomated Activity Cage LE 881 (Panlab) (45×45cm). This system detectsanimal movements from 2 levels of 4 side infrared sensors placed allaround the square cage. Mice were pretreated with the test compound(0.5-60 mg/kg po) and 30 min later, with Amph (2.5 mg/kg ip) or Amphjointly with CDP (3 mg/kg ip) and were placed individually in the motoractivity cages. The locomotor activity was measured in terms of totaldistance travelled (cm) over a period of 30 min. Each group consisted of8-10 mice.

Statistical analysis: a two-way analysis of variance (ANOVA) with Amphtreatment factor and drug treatment factor was performed. Bonferroni'sprocedure was used for multiple comparison post hoc analysis.D-amphetamine-chlordiazepoxide administration induced a significantincrease in locomotor activity.

Orally administered a ralfinamide (Ralf), manufactured according to theprocess of this invention, was found active in this experimental modelsignificantly decreasing the hyperactivity induced by Amph/ CDP mixture(FIG. 1).

1-58. (canceled)
 59. High purity(S)-2-[4-(3-fluorobenzyloxy)benzylamino]propanamide (safinamide) or(S)-2-[4-(2-fluorobenzyloxy)benzylamino]propanamide (ralfinamide) offormula (Ia) or (Ib)

and pharmaceutically acceptable acid salts thereof, produced by theprocess of: submitting a Schiff base intermediate respectively offormula (VIa) or (VIb)

to catalytic hydrogenation with hydrogen gas in the presence of aheterogeneous catalyst in a protic organic solvent and, when safinamideor ralfinamide are obtained in a free base form, optionally convertingsaid free base form to a salt thereof with a pharmaceutically acceptableacid.
 60. The product as in claim 59 wherein the catalytic hydrogenationis carried out by using an heterogeneous catalyst selected from nickel,rhodium, platinum and palladium catalysts on an inert support in thepresence of a solvent selected from lower aliphatic (C₁-C₅) alkanols.61. The product as in claim 59 wherein the catalyst is a palladium orplatinum catalyst.
 62. The product as in claim 59 wherein the catalystis wet 5% Pt/C (50% H₂O) or wet 10% Pd/C (50% H₂O).
 63. The product asin claim 59 wherein the pharmaceutically acceptable acid ismethanesulfonic acid.
 64. The product as in claim 59 wherein thehydrogen pressure is between 1 and 10 bars and the temperature isbetween 10° C. and 70° C.
 65. The product as in claim 64 wherein thehydrogen pressure is between 3 and 6 bars and the temperature is between25° C. and 40° C.
 66. The product as in claim 59 wherein the catalytichydrogenation is carried out on a Schiff base intermediate (VIa) or(VIb) which has been prepared through iminoalkylation of4-(3-fluorobenzyloxy)benzaldehyde (IVa) or4-(2-fluorobenzyloxy)benzaldehyde (IVb)

with L-alaninamide in the presence of a protic organic solvent.
 67. Theproduct as in claim 66 wherein the L-alaninamide is employed as an acidaddition salt thereof in the presence of a base in an amount sufficientto set free L-alaninamide from its salt.
 68. The product as in claim 66where the catalytic hydrogenation of the Schiff base intermediate isperformed on the same reaction mixture resulting from the completion ofthe iminoalkylation reaction under conditions which provoke theprecipitation of said Schiff base intermediate to obtain a suspension ofsaid intermediate in the same reaction solvent.
 69. The product as inclaim 66 wherein the Schiff base intermediate resulting from thecompletion of the iminoalkylation reaction is isolated before undergoingthe catalytic hydrogenation step.
 70. The product as in claim 66 whereinthe 4-(3-fluorobenzyloxy)benzaldehyde or4-(2-fluorobenzyloxy)benzaldehyde of formula (IVa) or (IVb) employed asthe starting material to obtain the Schiff base intermediate of formula(VIa) or (VIb) contains less than 0.03% (by weight), of the respectiveimpurities 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)benzaldehyde (Va) or3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)benzaldehyde (Vb)


71. The product according to claim 70 wherein the4-(3-fluorobenzyloxy)benzaldehyde (IVa) or4-(2-fluorobezyloxy)benzaldehyde (IVb) is obtained by alkylation of4-hydroxybenzaldehyde with, respectively, a 3-fluorobenzyl or2-fluorobenzyl derivative (IIIa) or (IIIb)

where Y is a leaving group selected from Cl, Br, I, OSO₂CH₃ andOSO₂-C₆H₄-pCH₃, in the presence of a base, and is submitted tocrystallization before the use in the successive reaction step.
 72. Theproduct as in claim 71 wherein Y is Cl.
 73. The product as in claim 71or 72 wherein the crystallization is carried out by adding an inertorganic non-solvent to a solution of the4-(3-fluorobenzyloxy)benzaldehyde (IVa) or4-(2-fluorobenzyloxy)benzaldehyde (IVb) in an inert organic solvent. 74.The product as in claim 73 wherein the inert organic non-solvent isselected from lower aliphatic hydrocarbons and the inert organic solventis selected from aromatic hydrocarbons.
 75. The product as in claim 74wherein the lower aliphatic hydrocarbon is n-hexane and the aromatichydrocarbon is toluene.
 76. The product as in any of claims 71 and 72wherein the crystallization is carried out by dissolving the4-(3-fluorobenzyloxy)benzaldehyde (IVa) or4-(2-fluorobenzyloxy)benzaldehyde (IVb) in a hot solvent selected fromcyclohexane, and a di(C3-C4)allcyl ether at reflux, and then cooling thesolution to a temperature at or below room temperature.
 77. The productas in any of claims 71 and 72 wherein the alkylation reaction is carriedout under phase transfer conditions.
 78. The product as in claim 77wherein the alkylation under phase transfer condition is performed in asolid/liquid system wherein the reagents and the phase transfer catalystare dissolved in a liquid organic phase and the solid phase isconstituted by an inorganic base or a salt of 4-hydroxy benzaldehydewith said inorganic base.
 79. The product as in claim 77 wherein thealkylation under phase transfer conditions is performed in aliquid/liquid system wherein the alkylating reagent 3-fluorobenzyl or2-fluorobenzyl derivative of formula (IIIa) or (IIIb) is dissolved in aliquid organic phase and the 4-hydroxybenzaldehyde is dissolved in anaqueous phase as a salt with an inorganic base.
 80. The product as inclaim 77 wherein the phase transfer catalyst is selected from quaternaryammonium or phosphonium salts or polyethyleneglycols of low molecularweight.
 81. The product of claim 80 wherein the amount of phase transfercatalyst employed is between 0.02 to 1 mole per mole of4-hydroxybenzaldheyde.
 82. The product as in claim 81 wherein the amountof phase-transfer catalyst is 0.1 to 1 mole per mole of4-hydroxybenzaldehyde.
 83. The product as in claim 78 wherein theorganic solvent of the liquid organic phase is selected from dialkylethers and aromatic hydrocarbons.
 84. The product as in claim 79 whereinthe molar ratio between the alkylating reagent of formula (IIIa) or(IIIb), and 4-hydroxybenzaldehyde is between 0.6 and 1.5.
 85. Theproduct as in claim 78 wherein the temperature is between 60° C. and160° C.
 86. The product as in claim 78 wherein the inorganic base isselected from Na₂CO₃, K₂CO₃, NaOH and KOH, the temperature is between80° C. and 120 ° C., and the ratio between the alkylating reagent offormula (IIIa) or (IIIb), and 4-hydroxybenzaldehyde is between 0.9 and1.1.
 87. The product as in claim 70 wherein safinamide or ralfinamide ortheir pharmaceutically acceptable acid salts, have a content of therespective impurity(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamide(IIa) or(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamide(IIb)

or their pharmaceutically acceptable acid salts, which is lower than0.03% (by weight).
 88. The product as in claim 87 wherein thepharmaceutically acceptable acid is methanesulfonic acid and the contentof the respective impurity of formula (IIa) or (IIb) as the salt withmethanesulfonic acid is lower than 0.01% (by weight).
 89. The product asin claim 61 wherein the catalyst is platinum.
 90. The product as inclaim 62 wherein the catalyst is wet 5% Pt/C (50% H₂O).
 91. The productas in claim 70 wherein the 4-(3-fluorobenzyloxy)benzaldehyde or4-(2-fluorobenzyloxy)benzaldehyde of formula (IVa) or (IVb) employed asthe starting material to obtain the Schiff base intermediate of formula(VIa) or (VIb) contains less than 0.01% (by weight), of the respectiveimpurities 3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy) benzaldehyde (Va) or3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy) benzaldehyde (Vb).
 92. Theproduct as in claim 76 wherein the hot solvent is diisopropylether. 93.The product as in claim 76 wherein the temperature is about 10-15° C.94. A method for treating epilepsy comprising administering to a patientin need thereof an effective amount of high purity safinamide or apharmaceutically acceptable acid salt thereof wherein the content of theimpurity(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamideof formula (IIa)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 95. A method for treating Parkinson's disease comprisingadministering to a patient in need thereof an effective amount of highpurity safinamide or a pharmaceutically acceptable acid salt thereofwherein the content of the impurity(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamideof formula (IIa)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 96. A method for treating Alzheimer's disease comprisingadministering to a patient in need thereof an effective amount of highpurity safinamide or a pharmaceutically acceptable acid salt thereofwherein the content of the impurity(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamideof formula (IIa)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 97. A method for treating depression comprisingadministering to a patient in need thereof an effective amount of highpurity safinamide or a pharmaceutically acceptable acid salt thereofwherein the content of the impurity(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamideof formula (IIa)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 98. A method for treating restless legs syndrome comprisingadministering to a patient in need thereof an effective amount of highpurity safinamide or a pharmaceutically acceptable acid salt thereofwherein the content of the impurity(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamideof formula (Ha)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 99. A method for treating migraine comprising administeringto a patient in need thereof an effective amount of high puritysafinamide or a pharmaceutically acceptable acid salt thereof whereinthe content of the impurity(S)-2-[3-(3-fluorobenzyl)-4-(3-fluorobenzyloxy)-benzylamino]propanamideof formula (IIa)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 100. A method for treating pain conditions comprisingadministering to a patient in need thereof an effective amount of highpurity ralfinamide or a salt thereof with a pharmaceutically acceptableacid wherein the content of the impurity(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideof formula (IIb)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 101. A method for treating migraine comprisingadministering to a patient in need thereof an effective amount of highpurity ralfinamide or a salt thereof with a pharmaceutically acceptableacid wherein the content of the impurity(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideof formula (IIb)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 102. A method for treating bipolar disorders comprisingadministering to a patient in need thereof an effective amount of highpurity ralfinamide or a salt thereof with a pharmaceutically acceptableacid wherein the content of the impurity(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideof formula (IIb)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 103. A method for treating depressions comprisingadministering to a patient in need thereof an effective amount of highpurity ralfinamide or a salt thereof with a pharmaceutically acceptableacid wherein the content of the impurity(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideof formula (IIb)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 104. A method for treating cardiovascular disorderscomprising administering to a patient in need thereof an effectiveamount of high purity ralfinamide or a salt thereof with apharmaceutically acceptable acid wherein the content of the impurity(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideof formula (IIb)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 105. A method for treating inflammatory disorderscomprising administering to a patient in need thereof an effectiveamount of high purity ralfinamide or a salt thereof with apharmaceutically acceptable acid wherein the content of the impurity(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideof formula (IIb)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 106. A method for treating urogenital disorders comprisingadministering to a patient in need thereof an effective amount of highpurity ralfinamide or a salt thereof with a pharmaceutically acceptableacid wherein the content of the impurity(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideof formula (IIb)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 107. A method for treating metabolic disorders comprisingadministering to a patient in need thereof an effective amount of highpurity ralfinamide or a salt thereof with a pharmaceutically acceptableacid wherein the content of the impurity(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideof formula (IIb)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).
 108. A method for treating gastrointestinal disorderscomprising administering to a patient in need thereof an effectiveamount of high purity ralfinamide or a salt thereof with apharmaceutically acceptable acid wherein the content of the impurity(S)-2-[3-(2-fluorobenzyl)-4-(2-fluorobenzyloxy)-benzylamino]propanamideof formula (IIb)

or a pharmaceutically acceptable acid salt thereof is lower than 0.03%(by weight).